Combination therapy with neoantigen vaccine

ABSTRACT

The present invention relates to neoplasia vaccine or immunogenic composition administered in combination with other agents, such as checkpoint blockade inhibitors for the treatment or prevention of neoplasia in a subject.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application claims benefit of and priority to U.S. Provisional Pat.Applications 61/919,576, filed Dec. 20, 2013 and 61/976,274, filed onApr. 7, 2014.

The foregoing applications, and all documents cited therein or duringtheir prosecution (“appln cited documents”) and all documents cited orreferenced in the appln cited documents, and all documents cited orreferenced herein (“herein cited documents”), and all documents cited orreferenced in herein cited documents, together with any manufacturer’sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated byreference herein, are hereby incorporated herein by reference, and maybe employed in the practice of the invention. More specifically, allreferenced documents are incorporated by reference to the same extent asif each individual document was specifically and individually indicatedto be incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to methods for the treatment of neoplasia,and more particularly tumors, by administering to a subject a neoplasiavaccine comprising a plurality of neoplasia/tumor specific neoantigensand at least one checkpoint inhibitor.

FEDERAL FUNDING LEGEND

This invention was made with government support under R01 CA155010-03awarded by NIH/NCI. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Approximately 1.6 million Americans are diagnosed with neoplasia everyyear, and approximately 580,000 people in the United States are expectedto die of the disease in 2013. Over the past few decades there beensignificant improvements in the detection, diagnosis, and treatment ofneoplasia, which have significantly increased the survival rate for manytypes of neoplasia. However, only about 60% of people diagnosed withneoplasia are still alive 5 years after the onset of treatment, whichmakes neoplasia the second leading cause of death in the United States.

Currently, there are a number of different existing cancer therapies,including ablation techniques (e.g., surgical procedures, cryogenic/heattreatment, ultrasound, radiofrequency, and radiation) and chemicaltechniques (e.g., pharmaceutical agents, cytotoxic/chemotherapeuticagents, monoclonal antibodies, and various combinations thereof).Unfortunately, such therapies are frequently associated with seriousrisk, toxic side effects, and extremely high costs, as well as uncertainefficacy.

There is a growing interest in cancer therapies that seek to targetcancerous cells with a patient’s own immune system (e.g., cancervaccines) because such therapies may mitigate/eliminate some of theherein-described disadvantages. Cancer vaccines are typically composedof tumor antigens and immunostimulatory molecules (e.g., cytokines orTLR ligands) that work together to induce antigen-specific cytotoxic Tcells that target and destroy tumor cells. Current cancer vaccinestypically contain shared tumor antigens, which are native proteins (i.e.-proteins encoded by the DNA of all the normal cells in the individual)that are selectively expressed or over-expressed in tumors found in manyindividuals. While such shared tumor antigens are useful in identifyingparticular types of tumors, they are not ideal as immunogens fortargeting a T-cell response to a particular tumor type because they aresubject to the immune dampening effects of self-tolerance. Vaccinescontaining tumor-specific and patient-specific neoantigens can overcomesome of the disadvantages of vaccines containing shared tumor antigens.

Described in the present application is combination therapy forachieving a desirable therapeutic result, and in particular in treatingcancer.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

The present invention relates to neoplasia vaccines or immunogeniccompositions administered in combination with one or more other agents,such as one or more checkpoint blockade inhibitors for the treatment orprevention of neoplasia in a subject.

In one aspect, the invention features a method of treating or preventinga neoplasia in a subject in need thereof that may comprise administeringto a subject in need thereof (a) a neoplasia vaccine or immunogeniccomposition; and (b) at least one checkpoint inhibitor. Theadministering can be serially or sequentially or at substantially thesame time or substantially simultaneously. For example, theadministering of the neoplasia vaccine or immunogenic composition andthe administering of the at least one checkpoint inhibitor can be atabout the same time or substantially simultaneously. Alternatively, theadministering of the neoplasia vaccine or immunogenic composition can beon one time schedule, e.g., weekly, biweekly, every three weeks,monthly, bimonthly, every quarter year (every three months), every thirdof a year (every four months), every five months, twice yearly (everysix months), every seven months, every eight months, every nine months,every ten months, every eleven months, annually or the like, and theadministering of the at least one checkpoint blockade inhibitor can beon a different schedule, typical for the checkpoint blockade inhibitorsuch that the subject or patient has two different treatment schedulesrunning concomitantly and the administering of the neoplasia vaccine orimmunogenic composition and the administering of the at least onecheckpoint inhibitor can be sequentially or serially. The neoplasiavaccine or immunogenic composition advantageously comprises at leastfour different neoantigens (and by different antigens it is intendedthat each antigen has a different neoepitope), e.g., at least 4 or 5 or6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30or 31 or 32 or 33 or 34 or 35 or 36 or 37 or 38 or 39 or 40 or moredifferent neoantigens can be in the neoplasia vaccine or immunogeniccomposition. The neoplasia vaccine or immunogenic composition can beadministered via subcompositions, each containing a portion of theneoantigens, and sub-compositions can be administered to differentplaces on the subject or patient, for instance, a composition comprising20 different neoantigens, can be administered in four (4)subcompositions, each containing 5 of the 20 different neoantigens, andthe four (4) subcompositions can be administered so as to endeavor todeliver each subcomposition at or near a draining lymph node of thepatient, e.g., to each of the arms and legs (e.g., thigh or upper thighor near buttocks or lower back on each side of the patient) so as toendeavor to deliver each subcomposition at or near a draining lymph nodeof the patient or subject. Of course, the number of locations and hencenumber of subcompositions can vary, e.g., the skilled practitioner couldconsider administration at or near the spleen to have a fifth point ofadministration, and the skilled practitioner can vary the locations suchthat only one, two or three are used (e.g., each arm and a leg, each oflegs and one arm, each of the legs and no arms, or only both arms). Thevaccine or immunogenic composition administered at the aforementionedvarious intervals can be different formulations, and the subcompositionsadministered at different places on the subject or patient during asingle administration can be different compositions. For instance, afirst administration can be of a whole antigen vaccine or immunogeniccomposition and a next or later administration can be of a vector (e.g.,viral vector or plasmid) that has expression of antigen(s) in vivo.Likewise, in the administration of different subcompositions todifferent locations on the patient or subject, some of thesubcompositions can comprise a whole antigen and some of thesubcompositions can comprise a vector (e.g., viral vector or plasmid)that has expression of antigen(s) in vivo. And some compositions andsubcompositions can comprise both vector(s) (e.g., viral vector orplasmid) that has / have expression of antigen(s) in vivo and wholeantigens. Some vectors (e.g., poxvirus) that have expression ofantigen(s) in vivo can have an immunostimulatory or adjuvanting effect,and hence compositions or subcompositions that contain such vectors canbe self-adjuvanting. Also, by changing up the nature of how the antigensare presented to the immune system, the administrations can “prime” andthen “boost” the immune system. And in this text, when there is mentionof a “vaccine” it is intended that the invention comprehends immunogeniccompositions, and when there is mention of a patient or subject it isintended that such an individual is a patient or subject in need of theherein disclosed treatments, administrations, compositions, andgenerally the subject invention.

In one embodiment, the neoplasia vaccine or immunogenic compositioncomprises at least two, at least three, at least four or at least fiveneoantigenic peptides. In another embodiment, the neoantigenic peptideranges from about 5 to about 50 amino acids in length. In anotherrelated embodiment, the neoantigenic peptide ranges from about 15 toabout 35 amino acids in length. Typically, the length is greater thanabout 15 or 20 amino acids, e.g., from 15 to 50 or about 75 amino acids.

In one embodiment, the neoplasia vaccine or immunogenic compositionfurther comprises a pH modifier and a pharmaceutically acceptablecarrier.

In one embodiment, the method further comprises administration of animmunomodulator or adjuvant (and hence the vaccine or immunogeniccomposition can include an immunomodulator or adjuvant). In anotherrelated embodiment, the immunomodulator or adjuvant is selected from thegroup consisting of poly-ICLC, 1018 ISS, aluminum salts, Amplivax. AS15,BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod,ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59,monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, MontanideISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PEPTEL,vector system, PLGA microparticles, resiquimod, SRL172, Virosomes andother Virus-like particles, YF-17D, VEGF trap, R848, beta-glucan,Pam3Cys, and Aquila’s QS21 stimulon. In another further embodiment, theimmunomodulator or adjuvant is poly-ICLC.

The dissolution of these polymers in water leads to an acid solutionwhich is neutralized, preferably to physiological pH, in order to givethe adjuvant solution into which the vaccine or immunogenic compositionor antigen(s) or vector(s) thereof is incorporated. The carboxyl groupsof the polymer are then partly in COO .

Preferably, a solution of adjuvant according to the invention,especially of carbomer, is prepared in distilled water, preferably inthe presence of sodium chloride, the solution obtained being at acidicpH. This stock solution is diluted by adding it to the required quantity(for obtaining the desired final concentration), or a substantial partthereof, of water charged with salt such as NaCl, preferablyphysiological saline (NaCl 9 g/l), all at once or in several portionswith concomitant or subsequent neutralization (pH 7.3 to 7.4),preferably with a base such as NaOH. This solution at physiological pHis used as is to reconstitute the vaccine, especially stored infreeze-dried or lyophilized form.

The polymer concentration in the final vaccine composition is 0.01% to2% w/v, more particularly 0.06 to 1% w/v, preferably 0.1 to 0.6% w/v.

Moreover, the invention applies to the use of any type of expressionvector, such as a viral expression vector, e.g., poxvirus (e.g.,orthopoxvirus or avipoxvirus such as vaccinia virus, including ModifiedVaccinia Ankara or MVA, MVA-BN, NYVAC according to WO-A-92/15672,fowlpox, e.g., TROVAX, canarypox, e.g., ALVAC (WO-A-95/27780 andWO-A-92/15672) pigeonpox, swinepox and the like), adenovirus, AAVherpesvirus, and lentivirus; or a plasmid or DNA or nucleic acidmolecule vector. Some vectors that arc cytoplasmic, such as poxvirusvectors, may be advantageous. However adenovirus, AAV and lentivirus canalso be advantageous to use in the practice of the invention.

In a ready-for-use, especially reconstituted, vaccine or immunogeniccomposition, the vector, e.g., viral vector, is present in thequantities within the ambit of the skilled person from this disclosureand the knowledge in the art (such as in patent and scientificliterature cited herein).

Whole antigen or vector, e.g., recombinant live vaccines generally existin a freeze-dried form allowing their storage and are reconstitutedimmediately before use in a solvent or excipient, which can include anadjuvant as herein discussed.

The subject of the invention is therefore also a vaccination orimmunization set or kit comprising, packaged separately, freeze-driedvaccine and a solution, advantageously including an adjuvant compound asherein discussed for the reconstitution of the freeze-dried vaccine.

The subject of the invention is also a method of vaccination orimmunization comprising or consisting essentially of or consisting ofadministering, e.g., by the parenteral, preferably subcutaneous,intramuscular or intradermal, route or by the mucosal route a vaccine orimmunogenic composition in accordance with the invention at the rate ofone or more administrations. Optionally this method includes apreliminary step of reconstituting the freeze-dried vaccine orimmunogenic composition (e.g., if lyophilized whole antigen or vector)in a solution, advantageously also including an adjuvant.

In one embodiment, the checkpoint inhibitor is an inhibitor of theprogrammed death-1 (PD-1) pathway. In another embodiment, the inhibitorof the PD-1 pathway is an anti-PD1 antibody. In a related embodiment,the inhibitor of the PD-1 pathway is Nivolumab.

In one embodiment, the checkpoint inhibitor is an anti-cytotoxicT-lymphocyte-associated antigen 4 (CTLA4) antibody. In a relatedembodiment, the anti-CTLA4 antibody is Ipilumumab or Tremelimumab.

In one embodiment, the subject is suffering from a neoplasia selectedfrom the group consisting of: Non-Hodgkin’s Lymphoma (NHL), clear cellRenal Cell Carcinoma (ccRCC), melanoma, sarcoma, leukemia or a cancer ofthe bladder, colon, brain, breast, head and neck, endometrium, lung,ovary, pancreas or prostate. In another embodiment, the neoplasia ismetastatic. In a further embodiment, the subject has no detectableneoplasia but is at high risk for disease recurrence. In a furtherrelated embodiment, the subject has previously undergone autologoushematopoietic stem cell transplant (AHSCT).

In one embodiment, administration of the checkpoint inhibitor isinitiated before initiation of administration of the neoplasia vaccineor immunogenic composition. In one embodiment, administration of thecheckpoint inhibitor is initiated after initiation of administration ofthe neoplasia vaccine or immunogenic composition. In one embodiment,administration of the checkpoint inhibitor is initiated simultaneouslywith the initiation of administration of the neoplasia vaccine orimmunogenic composition.

In another embodiment, administration of the checkpoint inhibitorcontinues every 2-8 or more weeks after the first administration of thecheckpoint inhibitor. In a further embodiment, administration of thecheckpoint inhibitor continues every 2, 3 or 4, 6 or 8 weeks after thefirst administration of the checkpoint inhibitor. In another furtherembodiment, the administration of the checkpoint inhibitor is withheldduring the week prior to administration of the neoplasia vaccine orimmunogenic composition. In still another further embodiment, theadministration of the checkpoint inhibitor is withheld duringadministration of the neoplasia vaccine or immunogenic composition.

In one embodiment, the administration of the checkpoint inhibitor isinitiated following tumor resection. In another embodiment,administration of the neoplasia vaccine or immunogenic composition isinitiated 1-15 weeks after tumor resection. In another furtherembodiment, administration of the neoplasia vaccine or immunogeniccomposition is initiated 4-12 weeks after tumor resection.

In one embodiment, administration of the neoplasia vaccine orimmunogenic composition is in a prime/ boost dosing regimen. In anotherembodiment, administration of the neoplasia vaccine or immunogeniccomposition is at weeks 1, 2, 3 or 4 as a prime. In another furtherembodiment, administration of the neoplasia vaccine or immunogeniccomposition is at months 2, 3, 4 or 5 as a boost.

In one embodiment, the vaccine or immunogenic composition isadministered at a dose of about 10 µg- 1 mg per 70 kg individual as toeach neoantigenic peptide. In another embodiment, the vaccine orimmunogenic composition is administered at an average weekly dose levelof about 10 µg- 2000 µg per 70 kg individual as to each neoantigenicpeptide. In another further embodiment, the checkpoint inhibitor isadministered at a dose of about 0.1-10 mg/kg. In another relatedembodiment, the administration is intravenous.

In one embodiment, the anti-CTLA4 antibody is administered at a dose ofabout 1 mg/kg- 3 mg/kg.

In one embodiment, the vaccine or immunogenic composition isadministered intravenously or subcutaneously.

In another embodiment, the checkpoint inhibitor is administeredintravenously or subcutaneously.

In another embodiment, the checkpoint inhibitor is administeredsubcutaneously within about 2 cm of the site of administration of theneoplasia vaccine or immunogenic composition.

In one embodiment, the checkpoint inhibitor is administered at a dose ofabout 0.1-1 mg per site of administration of the neoplasia vaccine orimmunogenic composition, per 70 kg individual.

In one embodiment, the method further comprises administration of one ormore additional agents. In another embodiment, the additional agents areselected from the group consisting of: chemotherapeutic agents,anti-angiogenesis agents and agents that reduce immune-suppression. In afurther embodiment, the one or more additional agents are one or moreanti-glucocorticoid induced tumor necrosis factor family receptor (GITR)agonistic antibodies.

In one embodiment the method may comprise administering the combinationtherapy within a standard of care for a particular cancer. In anotherembodiment the combination therapy is administered within a standard ofcare where addition of the combination therapy is synergistic with thesteps in the standard of care.

The invention comprehends performing methods as in U.S. Pat. ApplicationNo. 20110293637, incorporated herein by reference, e.g., a method ofidentifying a plurality of at least 4 subject-specific peptides andpreparing a subject-specific immunogenic composition that uponadministration presents the plurality of at least 4 subject-specificpeptides to the subject’s immune system, wherein the subject has a tumorand the subject-specific peptides are specific to the subject and thesubject’s tumor, said method comprising:

-   (i) identifying, including through    -   nucleic acid sequencing of a sample of the subject’s tumor and    -   nucleic acid sequencing of a non-tumor sample of the subject,

    a plurality of at least 4 tumor-specific non-silent mutations not    present in the non-tumor sample; and-   (ii) selecting from the identified non-silent mutations the    plurality of at least 4 subject-specific peptides, each having a    different tumor neo-epitope that is an epitope specific to the tumor    of the subject, from the identified plurality of tumor specific    mutations,    -   wherein each neo-epitope is an expression product of a        tumor-specific non-silent mutation not present in the non-tumor        sample, each neo-epitope binds to a HLA protein of the subject,        and selecting includes        -   determining binding of the subject-specific peptides to the            HLA protein, and-   (iii) formulating the subject-specific immunogenic composition for    administration to the subject so that upon administration the    plurality of at least 4 subject-specific peptides are presented to    the subject’s immune system,    -   wherein the selecting or formulating comprises at least one of:        -   including in the subject-specific immunogenic composition a            subject-specific peptide that includes an expression product            of an identified neo-ORF, wherein a neo-ORF is a            tumor-specific non-silent mutation not present in the            non-tumor sample that creates a new open reading frame, and        -   including in the subject-specific immunogenic composition a            subject-specific peptide that includes an expression product            of an identified point mutation and has a determined binding            to the HLA protein of the subject with an IC50 less than 500            nM,

whereby, the plurality of at least 4 subject-specific peptides areidentified, and the subject-specific immunogenic composition that uponadministration presents the plurality of at least 4 subject-specificpeptides to the subject’s immune system, wherein the subject-specificpeptides are specific to the subject and the subject’s tumor, isprepared; or a method of identifying a neoantigen comprising:

-   a. identifying a tumor specific mutation in an expressed gene of a    subject having cancer;-   b. wherein when said mutation identified in step (a) is a point    mutation:    -   i. identifying a mutant peptide having the mutation identified        in step (a), wherein said mutant peptide binds to a class I HLA        protein with a greater affinity than a wild-type peptide; and        has an IC50 less than 500 nrn;-   c. wherein when said mutation identified in step (a) is a    splice-site, frameshift, read-through or gene-fusion mutation:    -   i. identifying a mutant polypeptide encoded by the mutation        identified in step (a), wherein said mutant polypeptide binds to        a class I HLA protein; or a method of inducing a tumor specific        immune response in a subject comprising administering one or        more peptides or polypeptides identified and an adjuvant; or a        method of vaccinating or treating a subject for cancer        comprising:        -   a. identifying a plurality of tumor specific mutations in an            expressed gene of the subject wherein when said mutation            identified is a:            -   i. point mutation further identifying a mutant peptide                having the point mutation; and/or            -   ii. splice-site, frameshift, read-through or gene-fusion                mutation further identifying a mutant polypeptide                encoded by the mutation;        -   b. selecting one or more mutant peptides or polypeptides            identified in step (a) that binds to a class I HLA protein;        -   c. selecting the one or more mutant peptides or polypeptides            identified in step (b) that is capable of activating            anti-tumor CD8 T-cells; and        -   d. administering to the subject the one or more peptides or            polypeptides, autologous dendritic cells or antigen            presenting cells pulsed with the one or more peptides or            polypeptides selected in step (c); or preparing a            pharmaceutical composition comprising one identified            peptide(s), and performing method(s) as herein discussed.            Thus, the neoplasia vaccine or immunogenic composition            herein can be as in U.S. Pat. Application No. 20110293637.

Accordingly, it is an object of the invention to not encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) is provided by the Office upon request and payment ofthe necessary fee.

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings, incorporated herein by reference, wherein:

FIG. 1 depicts a flow process for making a personalized cancer vaccineor immunogenic composition.

FIG. 2 shows a flow process for pre-treatment steps for generating acancer vaccine or immunogenic composition for a melanoma patient.

FIG. 3 illustrates an immunization schedule based on a prime booststrategy according to an exemplary embodiment of the present invention.

FIG. 4 shows a time line indicating the primary immunological endpointaccording to an exemplary aspect of the invention.

FIG. 5 illustrates a time line for administering a co-therapy withcheckpoint blockade antibodies to evaluate the combination of relief oflocal immune suppression coupled with the stimulation of new immunityaccording to an exemplary embodiment of the invention. As shown in thescheme, patients who enter as appropriate candidates for checkpointblockade therapy, e.g., anti-PDL1 as shown here, may be entered andimmediately treated with antibody, while the vaccine or immunogeniccomposition is being prepared. Patients may then be vaccinated.Checkpoint blockade antibody dosing can be continued or possiblydeferred while the priming phase of vaccination occurs.

FIG. 6 shows a schematic depicting drug product processing of individualneoantigenic peptides into pools of 4 subgroups according to anexemplary embodiment of the invention.

FIG. 7 shows the results of quantitative PCR to assess the levels ofinduction of a number of key immune markers after stimulation of mousedendritic cells using a neoantigenic formulation.

FIG. 8 is a schematic outline of study 1: Nivolumab vs Nivolumab andNeoVax following AHSCT in Non-Hodgkin’s Lymphoma.

FIG. 9 is a schematic outline of study 2: Nivolumab and NeoVax inmetastatic melanoma and metastatic RCC.

FIGS. 10A-D are schematics of four studies. (A) Study 3a shows doseescalation of reduced intensity intravenous Ipilimumab in metastaticmelanoma. (B) Study 3b shows dose escalation of sub-cutaneous Ipilimumab(Local) in metastatic melanoma. (C) Study 3c shows dose escalation ofreduced intensity intravenous Ipilimumab in high-risk Melanoma. (D)Study 3d shows dose escalation of sub-cutaneous Ipilimumab (Local) inhigh-risk melanoma.

FIG. 11 illustrates a study scheme combining NeoVax and Ipilimumab totreat high-risk renal cell carcinoma.

FIG. 12 illustrates a treatment scheme for a study combining NeoVax andIpilimumab to treat high-risk renal cell carcinoma.

DETAILED DESCRIPTION OF THE INVENTION Definitions

To facilitate an understanding of the present invention, a number ofterms and phrases are defined herein:

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of thestated value. Unless otherwise clear from context, all numerical valuesprovided herein arc modified by the term about.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. Unless specifically stated orobvious from context, as used herein, the terms “a,” “an,” and “the” areunderstood to be singular or plural.

By “agent” is meant any small molecule chemical compound, antibody,nucleic acid molecule, or polypeptide, or fragments thereof.

Immune checkpoints are inhibitory pathways that slow down or stop immunereactions and prevent excessive tissue damage from uncontrolled activityof immune cells. By “checkpoint inhibitor” is meant to refer to anysmall molecule chemical compound, antibody, nucleic acid molecule, orpolypeptide, or fragments thereof, that inhibits the inhibitorypathways, allowing more extensive immune activity. In certainembodiments, the checkpoint inhibitor is an inhibitor of the programmeddeath-1 (PD-1) pathway, for example an anti-PD1 antibody, such as, butnot limited to Nivolumab. In other embodiments, the checkpoint inhibitoris an anti-cytotoxic T-lymphocyte-associated antigen (CTLA-4) antibody.In additional embodiments, the checkpoint inhibitor is targeted atanother member of the CD28CTLA4 Ig superfamily such as BTLA, LAG3, ICOS,PDL1 or KIR Page et al., Annual Review of Medicine 65:27 (2014)). Infurther additional embodiments, the checkpoint inhibitor is targeted ata member of the TNFR superfamily such as CD40, OX40, CD137, GITR, CD27or TIM-3. In some cases targeting a checkpoint inhibitor is accomplishedwith an inhibitory antibody or similar molecule. In other cases, it isaccomplished with an agonist for the target; examples of this classinclude the stimulatory targets OX40 and GITR.

The term “combination” embraces the administration of a neoplasiavaccine or immunogenic composition (e.g. a pooled sample ofneoplasia/tumor specific neoantigens) and one or more checkpointinhibitors, as part of a treatment regimen intended to provide abeneficial (additive or synergistic) effect from the co-action of one ormore of these therapeutic agents. The combination may also include oneor more additional agents, for example, but not limited to,chemotherapeutic agents, anti-angiogenesis agents and agents that reduceimmune-suppression. The beneficial effect of the combination includes,but is not limited to, pharmacokinetic or pharmacodynamic co-actionresulting from the combination of therapeutic agents. Administration ofthese therapeutic agents in combination typically is carried out over adefined time period (for example, minutes, hours, days, or weeksdepending upon the combination selected).

“Combination therapy” is intended to embrace administration of thesetherapeutic agents in a sequential manner, that is, wherein eachtherapeutic agent is administered at a different time, as well asadministration of these therapeutic agents, or at least two of thetherapeutic agents, in a substantially simultaneous manner.Substantially simultaneous administration can be accomplished, forexample, by administering to the subject a single capsule having a fixedratio of each therapeutic agent or in multiple, single capsules for eachof the therapeutic agents. For example, one combination of the presentinvention may comprise a pooled sample of tumor specific neoantigens anda checkpoint inhibitor administered at the same or different times, orthey can be formulated as a single, co-formulated pharmaceuticalcomposition comprising the two compounds. As another example, acombination of the present invention (e.g., a pooled sample of tumorspecific neoantigens and a checkpoint inhibitor and/or an anti-CTLA4antibody) may be formulated as separate pharmaceutical compositions thatcan be administered at the same or different time. As used herein, theterm “simultaneously” is meant to refer to administration of one or moreagents at the same time. For example, in certain embodiments, aneoplasia vaccine or immunogenic composition and a checkpoint inhibitorare administered simultaneously. Simultaneously includes administrationcontemporaneously, that is during the same period of time. In certainembodiments, the one or more agents are administered simultaneously inthe same hour, or simultaneously in the same day. Sequential orsubstantially simultaneous administration of each therapeutic agent canbe effected by any appropriate route including, but not limited to, oralroutes, intravenous routes, sub-cutaneous routes, intramuscular routes,direct absorption through mucous membrane tissues (e.g., nasal, mouth,vaginal, and rectal), and ocular routes (e.g., intravitreal,intraocular, etc.). The therapeutic agents can be administered by thesame route or by different routes. For example, one component of aparticular combination may be administered by intravenous injectionwhile the other component(s) of the combination may be administeredorally. The components may be administered in any therapeuticallyeffective sequence. The phrase “combination” embraces groups ofcompounds or non-drug therapies useful as part of a combination therapy.

The term “ncoantigcn” or “ncoantigcnic” means a class of tumor antigensthat arises from a tumor-specific mutation(s) which alters the aminoacid sequence of genome encoded proteins.

By “neoplasia” is meant any disease that is caused by or results ininappropriately high levels of cell division, inappropriately low levelsof apoptosis, or both. For example, cancer is an example of a neoplasia.Examples of cancers include, without limitation, leukemia (e.g., acuteleukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acutemyeloblastic leukemia, acute promyelocytic leukemia, acutemyelomonocytic leukemia, acute monocytic leukemia, acuteerythroleukemia, chronic leukemia, chronic myelocytic leukemia, chroniclymphocytic leukemia), polycythemia vera, lymphoma (e.g., Hodgkin’sdisease, non-Hodgkin’s disease), Waldenstrom’s macroglobulinemia, heavychain disease, and solid tumors such as sarcomas and carcinomas (e.g.,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing’s tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm’s tumor, cervical cancer, uterinecancer, testicular cancer, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma,meningioma, melanoma, neuroblastoma, and retinoblastoma).Lymphoproliferative disorders are also considered to be proliferativediseases.

The term “neoplasia vaccine” is meant to refer to a pooled sample ofneoplasia/tumor specific neoantigens, for example at least two, at leastthree, at least four, at least five, or more neoantigenic peptides. A“vaccine” is to be understood as meaning a composition for generatingimmunity for the prophylaxis and/or treatment of diseases (e.g.,neoplasia/tumor). Accordingly, vaccines are medicaments which compriseantigens and are intended to be used in humans or animals for generatingspecific defense and protective substance by vaccination. A “neoplasiavaccine composition ” can include a pharmaceutically acceptableexcipient, carrier or diluent.

The term “pharmaceutically acceptable” refers to approved or approvablcby a regulatory agency of the Federal or a state government or listed inthe U.S. Pharmacopeia or other generally recognized pharmacopeia for usein animals, including humans.

A “pharmaceutically acceptable excipient, carrier or diluent” refers toan excipient, carrier or diluent that can be administered to a subject,together with an agent, and which does not destroy the pharmacologicalactivity thereof and is nontoxic when administered in doses sufficientto deliver a therapeutic amount of the agent.

A “pharmaceutically acceptable salt” of pooled tumor specificneoantigens as recited herein may be an acid or base salt that isgenerally considered in the art to be suitable for use in contact withthe tissues of human beings or animals without excessive toxicity,irritation, allergic response, or other problem or complication. Suchsalts include mineral and organic acid salts of basic residues such asamines, as well as alkali or organic salts of acidic residues such ascarboxylic acids. Specific pharmaceutical salts include, but are notlimited to, salts of acids such as hydrochloric, phosphoric,hydrobromic, malic, glycolic, fumaric, sulfuric, sulfamic, sulfanilic,formic, toluenesulfonic, methanesulfonic, benzene sulfonic, ethanedisulfonic, 2-hydroxyethylsulfonic, nitric, benzoic, 2-acetoxybenzoic,citric, tartaric, lactic, stearic, salicylic, glutamic, ascorbic,pamoic, succinic, fumaric, maleic, propionic, hydroxymalcic, hydroiodic,phenylacetic, alkanoic such as acetic, HOOC—(CH2)n—COOH where n is 0-4,and the like. Similarly, pharmaceutically acceptable cations include,but are not limited to sodium, potassium, calcium, aluminum, lithium andammonium. Those of ordinary skill in the art will recognize from thisdisclosure and the knowledge in the art that further pharmaceuticallyacceptable salts for the pooled tumor specific neoantigens providedherein, including those listed by Remington’s Pharmaceutical Sciences,17th ed., Mack Publishing Company, Easton, PA, p. 1418 (1985). Ingeneral, a pharmaceutically acceptable acid or base salt can besynthesized from a parent compound that contains a basic or acidicmoiety by any conventional chemical method. Briefly, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in anappropriate solvent.

By a “polypeptide” or “peptide” is meant a polypeptide that has beenseparated from components that naturally accompany it. Typically, thepolypeptide is isolated when it is at least 60%, by weight, free fromthe proteins and naturally-occurring organic molecules with which it isnaturally associated. Preferably, the preparation is at least 75%, morepreferably at least 90%, and most preferably at least 99%, by weight, apolypeptide. An isolated polypeptide may be obtained, for example, byextraction from a natural source, by expression of a recombinant nucleicacid encoding such a polypeptide; or by chemically synthesizing theprotein. Purity can be measured by any appropriate method, for example,column chromatography, polyacrylamide gel electrophoresis, or by HPLCanalysis.

As used herein, the terms “prevent,” “preventing,” “prevention,”“prophylactic treatment,” and the like, refer to reducing theprobability of developing a disease or condition in a subject, who doesnot have, but is at risk of or susceptible to developing a disease orcondition.

The term “prime/ boost” or “prime/ boost dosing regimen” is meant torefer to the successive administrations of a vaccine or immunogenic orimmunological compositions. The priming administration (priming) is theadministration of a first vaccine or immunogenic or immunologicalcomposition type and may comprise one, two or more administrations. Theboost administration is the second administration of a vaccine orimmunogenic or immunological composition type and may comprise one, twoor more administrations, and, for instance, may comprise or consistessentially of annual administrations. In certain embodiments,administration of the neoplasia vaccine or immunogenic composition is ina prime/ boost dosing regimen.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50,as well as all intervening decimal values between the aforementionedintegers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,and 1.9. With respect to sub-ranges, “nested sub-ranges” that extendfrom either end point of the range are specifically contemplated. Forexample, a nested sub-range of an exemplary range of 1 to 50 maycomprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

A “receptor” is to be understood as meaning a biological molecule or amolecule grouping capable of binding a ligand. A receptor may serve, totransmit information in a cell, a cell formation or an organism. Thereceptor comprises at least one receptor unit and frequently containstwo or more receptor units, where each receptor unit may consist of aprotein molecule, in particular a glycoprotein molecule. The receptorhas a structure that complements the structure of a ligand and maycomplex the ligand as a binding partner. Signaling information may betransmitted by conformational changes of the receptor following bindingwith the ligand on the surface of a cell. According to the invention, areceptor may refer to particular proteins of MHC classes I and IIcapable of forming a receptor/ligand complex with a ligand, inparticular a peptide or peptide fragment of suitable length.

The term “subject” refers to an animal which is the object of treatment,observation, or experiment. By way of example only, a subject includes,but is not limited to, a mammal, including, but not limited to, a humanor a non-human mammal, such as a non-human primate, bovine, equine,canine, ovine, or feline.

The terms “treat,” “treated,” “treating,” “treatment,” and the like aremeant to refer to reducing or ameliorating a disorder and/or symptomsassociated therewith (e.g., a neoplasia or tumor). “Treating” may referto administration of the combination therapy to a subject after theonset, or suspected onset, of a cancer. “Treating” includes the conceptsof “alleviating”, which refers to lessening the frequency of occurrenceor recurrence, or the severity, of any symptoms or other ill effectsrelated to a cancer and/or the side effects associated with cancertherapy. The term “treating” also encompasses the concept of “managing”which refers to reducing the severity of a particular disease ordisorder in a patient or delaying its recurrence, e.g., lengthening theperiod of remission in a patient who had suffered from the disease. Itis appreciated that, although not precluded, treating a disorder orcondition does not require that the disorder, condition, or symptomsassociated therewith be completely eliminated.

The term “therapeutic effect” refers to some extent of relief of one ormore of the symptoms of a disorder (e.g., a neoplasia or tumor) or itsassociated pathology. “Therapeutically effective amount” as used hereinrefers to an amount of an agent which is effective, upon single ormultiple dose administration to the cell or subject, in prolonging thesurvivability of the patient with such a disorder, reducing one or moresigns or symptoms of the disorder, preventing or delaying, and the likebeyond that expected in the absence of such treatment. “Therapeuticallyeffective amount” is intended to qualify the amount required to achievea therapeutic effect. A physician or veterinarian having ordinary skillin the art can readily determine and prescribe the “therapeuticallyeffective amount” (e.g., ED50) of the pharmaceutical compositionrequired. For example, the physician or veterinarian could start dosesof the compounds of the invention employed in a pharmaceuticalcomposition at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

The combination therapy disclosed herein constitutes a new method fortreating various types of cancer. The combination therapy describedherein also provides a method of therapy for achieving clinical benefitwithout an unacceptable level of side effects.

The present invention relates to methods for the treatment of neoplasia,and more particularly tumors, by administering to a subject a neoplasiavaccine or immunogenic composition comprising a plurality ofneoplasia/tumor specific neoantigens and at least one checkpointinhibitor.

As described in more detail herein, whole genome/exome sequencing may beused to identify all, or nearly all, mutated neoantigens that areuniquely present in a neoplasia/tumor of an individual patient, and thatthis collection of mutated neoantigens may be analyzed to identify aspecific, optimized subset of neoantigens for use as a personalizedcancer vaccine or immunogenic composition for treatment of the patient’sneoplasia/tumor. For example, a population of neoplasia/tumor specificneoantigens may be identified by sequencing the neoplasia/tumor andnormal DNA of each patient to identify tumor-specific mutations, and thepatient’s HLA allotype can be identified. The population ofneoplasia/tumor specific neoantigens and their cognate native antigensmay then be subject to bioinformatic analysis using validated algorithmsto predict which tumor-specific mutations create epitopes that couldbind to the patient’s HLA allotype. Based on this analysis, a pluralityof peptides corresponding to a subset of these mutations may be designedand synthesized for each patient, and pooled together for use as acancer vaccine or immunogenic composition in immunizing the patient.

The immune system can be classified into two functional subsystems: theinnate and the acquired immune system. The innate immune system is thefirst line of defense against infections, and most potential pathogensare rapidly neutralized by this system before they can cause, forexample, a noticeable infection. The acquired immune system reacts tomolecular structures, referred to as antigens, of the intrudingorganism. There are two types of acquired immune reactions, whichinclude the humoral immune reaction and the cell-mediated immunereaction. In the humoral immune reaction, antibodies secreted by B cellsinto bodily fluids bind to pathogen-derived antigens, leading to theelimination of the pathogen through a variety of mechanisms, e.g.complement-mediated lysis. In the cell-mediated immune reaction, T-cellscapable of destroying other cells are activated. For example, ifproteins associated with a disease are present in a cell, they arefragmented proteolytically to peptides within the cell. Specific cellproteins then attach themselves to the antigen or peptide formed in thismanner and transport them to the surface of the cell, where they arepresented to the molecular defense mechanisms, in particular T-cells, ofthe body. Cytotoxic T cells recognize these antigens and kill the cellsthat harbor the antigens.

The molecules that transport and present peptides on the cell surfaceare referred to as proteins of the major histocompatibility complex(MHC). MHC proteins are classified into two types, referred to as MHCclass I and MHC class II. The structures of the proteins of the two MHCclasses are very similar; however, they have very different functions.Proteins of MHC class I are present on the surface of almost all cellsof the body, including most tumor cells. MHC class I proteins are loadedwith antigens that usually originate from endogenous proteins or frompathogens present inside cells, and are then presented to naïve orcytotoxic T-lymphocytes (CTLs). MHC class II proteins are present ondendritic cells, B- lymphocytes, macrophages and otherantigen-presenting cells. They mainly present peptides, which areprocessed from external antigen sources, i.e. outside of the cells, toT-helper (Th) cells. Most of the peptides bound by the MHC class Iproteins originate from cytoplasmic proteins produced in the healthyhost cells of an organism itself, and do not normally stimulate animmune reaction. Accordingly, cytotoxic T-lymphocytes that recognizesuch self-peptide-presenting MHC molecules of class I are deleted in thethymus (central tolerance) or, after their release from the thymus, aredeleted or inactivated, i.e. tolerized (peripheral tolerance). MHCmolecules are capable of stimulating an immune reaction when theypresent peptides to non-tolerized T-lymphocytes. Cytotoxic T-lymphocyteshave both T-cell receptors (TCR) and CD8 molecules on their surface.T-Cell receptors are capable of recognizing and binding peptidescomplexed with the molecules of MHC class I. Each cytotoxic T-lymphocyteexpresses a unique T-cell receptor which is capable of binding specificMHC/peptide complexes.

The peptide antigens attach themselves to the molecules of MHC class Iby competitive affinity binding within the endoplasmic reticulum, beforethey are presented on the cell surface. Here, the affinity of anindividual peptide antigen is directly linked to its amino acid sequenceand the presence of specific binding motifs in defined positions withinthe amino acid sequence. If the sequence of such a peptide is known, itis possible to manipulate the immune system against diseased cellsusing, for example, peptide vaccines.

One of the critical barriers to developing curative and tumor-specificimmunotherapy is the identification and selection of highly specific andrestricted tumor antigens to avoid autoimmunity. Tumor neoantigens,which arise as a result of genetic change (e.g., inversions,translocations, deletions, missense mutations, splice site mutations,etc.) within malignant cells, represent the most tumor-specific class ofantigens. Neoantigens have rarely been used in cancer vaccine orimmunogenic compositions due to technical difficulties in identifyingthem, selecting optimized neoantigens, and producing neoantigens for usein a vaccine or immunogenic composition. These problems may be addressedby:

-   identifying all, or nearly all, mutations in the neoplasia/tumor at    the DNA level using whole genome, whole exome (e.g., only captured    exons), or RNA sequencing of tumor versus matched germline samples    from each patient;-   analyzing the identified mutations with one or more peptide-MHC    binding prediction algorithms to generate a plurality of candidate    neoantigen T cell epitopes that are expressed within the    neoplasia/tumor and may bind patient HLA alleles; and-   synthesizing the plurality of candidate neoantigen peptides selected    from the sets of all neoORF peptides and predicted binding peptides    for use in a cancer vaccine or immunogenic composition.

As described herein, there is a large body of evidence in both animalsand humans that mutated epitopes are effective in inducing an immuneresponse and that cases of spontaneous tumor regression or long termsurvival correlate with CD8+ T-cell responses to mutated epitopes(Buckwalter and Srivastava PK. “It is the antigen(s), stupid” and otherlessons from over a decade of vaccitherapy of human cancer. Seminars inimmunology 20:296-300 (2008); Karanikas et al, High frequency ofcytolytic T lymphocytes directed against a tumor-specific mutatedantigen detectable with HLA tetramers in the blood of a lung carcinomapatient with long survival. Cancer Res. 61:3718-3724 (2001); Lennerz etal, The response of autologous T cells to a human melanoma is dominatedby mutated neoantigens. Proc Natl Acad Sci U S A.102:16013 (2005)) andthat “immunoediting” can be tracked to alterations in expression ofdominant mutated antigens in mice and man (Matsushita et al, Cancerexome analysis reveals a T-cell-dependent mechanism of cancerimmunoediting Nature 482:400 (2012); DuPage et al, Expression oftumor-specific antigens underlies cancer immunoediting Nature 482:405(2012); and Sampson et al, Immunologic escape after prolongedprogression-free survival with epidermal growth factor receptor variantIII peptide vaccination in patients with newly diagnosed glioblastoma JClin Oncol. 28:4722-4729 (2010)). In one embodiment, the mutatedepitopes of a cancer patient are determined.

In one embodiment mutated epitopes arc determined by sequencing thegenome and/or exome of tumor tissue and healthy tissue from a cancerpatient using next generation sequencing technologies. In anotherembodiment genes that are selected based on their frequency of mutationand ability to act as a neoantigen are sequenced using next generationsequencing technology. Next-generation sequencing applies to genomesequencing, genome resequencing, transcriptome profiling (RNA-Seq),DNA-protein interactions (ChIP-sequencing), and epigenomecharacterization (de Magalhães JP, Finch CE, Janssens G (2010).“Next-generation sequencing in aging research: emerging applications,problems, pitfalls and possible solutions”. Ageing Research Reviews 9(3): 315-323; Hall N (May 2007). “Advanced sequencing technologies andtheir wider impact in microbiology”. J. Exp. Biol. 209 (Pt 9):1518-1525; Church GM (January 2006). “Genomes for all”. Sci. Am. 294(1): 46-54; ten Bosch JR, Grody WW (2008). “Keeping Up with the NextGeneration”. The Journal of Molecular Diagnostics 10 (6): 484-492;Tucker T, Marra M, Friedman JM (2009). “Massively Parallel Sequencing:The Next Big Thing in Genetic Medicine”. The American Journal of HumanGenetics 85 (2): 142-154). Next-generation sequencing can now rapidlyreveal the presence of discrete mutations such as coding mutations inindividual tumors, most commonly single amino acid changes (e.g.,missense mutations) and less frequently novel stretches of amino acidsgenerated by frame-shift insertions/deletions/gene fusions, read-throughmutations in stop codons, and translation of improperly spliced introns(e.g., neoORFs). NeoORFs are particularly valuable as immunogens becausethe entirety of their sequence is completely novel to the immune systemand so are analogous to a viral or bacterial foreign antigen. Thus,ncoORFs: (1) are highly specific to the tumor (i.e. there is noexpression in any normal cells); (2) can bypass central tolerance,thereby increasing the precursor frequency of neoantigen-specific CTLs.For example, the power of utilizing analogous foreign sequences in atherapeutic anti-cancer vaccine or immunogenic composition was recentlydemonstrated with peptides derived from human papilloma virus (HPV).~50% of the 19 patients with pre-neoplastic, viral-induced disease whoreceived 3-4 vaccinations of a mix of HPV peptides derived from theviral oncogenes E6 and E7 maintained a complete response for ≥24 months(Kenter et a, Vaccination against HPV-16 Oncoproteins for VulvarIntraepithelial Neoplasia NEJM 361:1838 (2009)).

Sequencing technology has revealed that each tumor contains multiple,patient-specific mutations that alter the protein coding content of agene. Such mutations create altered proteins, ranging from single aminoacid changes (caused by missense mutations) to addition of long regionsof novel amino acid sequence due to frame shifts, read-through oftermination codons or translation of intron regions (novel open readingframe mutations; neoORFs). These mutated proteins are valuable targetsfor the host’s immune response to the tumor as, unlike native proteins,they are not subject to the immune-dampening effects of self-tolerance.Therefore, mutated proteins are more likely to be immunogenic and arealso more specific for the tumor cells compared to normal cells of thepatient.

An alternative method for identifying tumor specific neoantigens isdirect protein sequencing. Protein sequencing of enzymatic digests usingmultidimensional MS techniques (MSn) including tandem mass spectrometry(MS/MS)) can also be used to identify neoantigens of the invention. Suchproteomic approaches permit rapid, highly automated analysis (see, e.g.,K. Gcvaert and J. Vandekerckhove, Electrophoresis 21:1145-1154 (2000)).It is further contemplated within the scope of the invention thathigh-throughput methods for de novo sequencing of unknown proteins maybe used to analyze the proteome of a patient’s tumor to identifyexpressed neoantigens. For example, meta shotgun protein sequencing maybe used to identify expressed neoantigens (see e.g., Guthals et al.(2012) Shotgun Protein Sequencing with Meta-contig Assembly, Molecularand Cellular Proteomics 11(10):1084-96).

Tumor specific neoantigens may also be identified using MHC multimers toidentify neoantigen-specific T-cell responses. For example,high-throughput analysis of neoantigen-specific T-cell responses inpatient samples may be performed using MHC tetramer-based screeningtechniques (see e.g., Hombrink et al. (2011) High-ThroughputIdentification of Potential Minor Histocompatibility Antigens by MHCTetramer-Based Screening: Feasibility and Limitations 6(8):1-11; Hadrupet al. (2009) Parallel detection of antigen-specific T-cell responses bymultidimensional encoding of MHC multimers, Nature Methods, 6(7):520-26;van Rooij et al. (2013) Tumor exome analysis reveals neoantigen-specificT-cell reactivity in an Ipilimumab-responsive melanoma, Journal ofClinical Oncology, 31:1-4; and Heemskerk et al. (2013) The cancerantigenome, EMBO Journal, 32(2):194-203). Such tetramer-based screeningtechniques may be used for the initial identification of tumor specificneoantigens, or alternatively as a secondary screening protocol toassess what neoantigens a patient may have already been exposed to,thereby facilitating the selection of candidate neoantigens for theinvention.

In one embodiment the sequencing data derived from determining thepresence of mutations in a cancer patient is analysed to predictpersonal mutated peptides that can bind to HLA molecules of theindividual. In one embodiment the data is analysed using a computer. Inanother embodiment the sequence data is analysed for the presence ofneoantigens. In one embodiment neoantigens are determined by theiraffinity to MHC molecules. Efficiently choosing which particularmutations to utilize as immunogen requires identification of the patientHLA type and the ability to predict which mutated peptides wouldefficiently bind to the patient’s HLA alleles. Recently, neural networkbased learning approaches with validated binding and non-bindingpeptides have advanced the accuracy of prediction algorithms for themajor HLA-A and -B alleles. Utilizing the recently improved algorithmsfor predicting which missense mutations create strong binding peptidesto the patient’s cognate MHC molecules, a set of peptides representativeof optimal mutated epitopes (both neoORF and missense) for each patientmay be identified and prioritized (Zhang et al, Machine learningcompetition in immunology ... Prediction of HLA class I binding peptidesJ Immunol Methods 374:1 (2011); Lundegaard et al Prediction of epitopesusing neural network based methods J Immunol Methods 374:26 (2011)).

Targeting as many mutated epitopes as practically possible takesadvantage of the enormous capacity of the immune system, prevents theopportunity for immunological escape by down-modulation of a particularimmune targeted gene product, and compensates for the known inaccuracyof epitope prediction approaches. Synthetic peptides provide aparticularly useful means to prepare multiple immunogens efficiently andto rapidly translate identification of mutant epitopes to an effectivevaccine or immunogenic composition. Peptides can be readily synthesizedchemically and easily purified utilizing reagents free of contaminatingbacteria or animal substances. The small size allows a clear focus onthe mutated region of the protein and also reduces irrelevant antigeniccompetition from other components (unmutated protein or viral vectorantigens).

In one embodiment the drug formulation is a multi-epitope vaccine orimmunogenic composition of long peptides. Such “long” peptides undergoefficient internalization, processing and cross-presentation inprofessional antigen-presenting cells such as dendritic cells, and havebeen shown to induce CTLs in humans (Melief and van der Burg,Immunotherapy of established (pre) malignant disease by synthetic longpeptide vaccines Nature Rev Cancer 8:351 (2008)). In one embodiment atleast 1 peptide is prepared for immunization. In a prefered embodiment20 or more peptides are prepared for immunization. In one embodiment theneoantigenic peptide ranges from about 5 to about 50 amino acids inlength. In another embodiment peptides from about 15 to about 35 aminoacids in length is synthesized. In prefered embodiment the neoantigenicpeptide ranges from about 20 to about 35 amino acids in length.

Production of Tumor Specific Neoantigens

The present invention is based, at least in part, on the ability topresent the immune system of the patient with a pool of tumor specificneoantigens. One of skill in the art from this disclosure and theknowledge in the art will appreciate that there are a variety of ways inwhich to produce such tumor specific neoantigens. In general, such tumorspecific neoantigens may be produced either in vitro or in vivo. Tumorspecific neoantigens may be produced in vitro as peptides orpolypeptides, which may then be formulated into a personalized neoplasiavaccine or immunogenic composition and administered to a subject. Asdescribed in further detail herein, such in vitro production may occurby a variety of methods known to one of skill in the art such as, forexample, peptide synthesis or expression of a peptide/polypeptide from aDNA or RNA molecule in any of a variety of bacterial, eukaryotic, orviral recombinant expression systems, followed by purification of theexpressed peptide/polypeptide. Alternatively, tumor specific neoantigensmay be produced in vivo by introducing molecules (e.g., DNA, RNA, viralexpression systems, and the like) that encode tumor specific neoantigensinto a subject, whereupon the encoded tumor specific neoantigens areexpressed. The methods of in vitro and in vivo production of neoantigensis also further described herein as it relates to pharmaceuticalcompositions and methods of delivery of the combination therapy.

In Vitro Peptide/Polypeptide Synthesis

Proteins or peptides may be made by any technique known to those ofskill in the art, including the expression of proteins, polypeptides orpeptides through standard molecular biological techniques, the isolationof proteins or peptides from natural sources, in vitro translation, orthe chemical synthesis of proteins or peptides. The nucleotide andprotein, polypeptide and peptide sequences corresponding to variousgenes have been previously disclosed, and may be found at computerizeddatabases known to those of ordinary skill in the art. One such databaseis the National Center for Biotechnology Information’s Genbank andGenPept databases located at the National Institutes of Health website.The coding regions for known genes may be amplified and/or expressedusing the techniques disclosed herein or as would be known to those ofordinary skill in the art. Alternatively, various commercialpreparations of proteins, polypeptides and peptides are known to thoseof skill in the art.

Peptides can be readily synthesized chemically utilizing reagents thatare free of contaminating bacterial or animal substances (Merrifield RB:Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J.Am. Chem. Soc. 85:2149-54, 1963). In certain embodiments, neoantigenicpeptides are prepared by (1) parallel solid-phase synthesis onmulti-channel instruments using uniform synthesis and cleavageconditions; (2) purification over a RP-HPLC column with columnstripping; and re-washing, but not replacement, between peptides;followed by (3) analysis with a limited set of the most informativeassays. The Good Manufacturing Practices (GMP) footprint can be definedaround the set of peptides for an individual patient, thus requiringsuite changeover procedures only between syntheses of peptides fordifferent patients.

Alternatively, a nucleic acid (e.g., a polynucleotide) encoding aneoantigenic peptide of the invention may be used to produce theneoantigenic peptide in vitro. The polynucleotide may be, e.g., DNA,cDNA, PNA, CNA, RNA, either single- and/or double-stranded, or native orstabilized forms of polynucleotides, such as e.g. polynucleotides with aphosphorothiate backbone, or combinations thereof and it may or may notcontain introns so long as it codes for the peptide. In one embodimentin vitro translation is used to produce the peptide. Many exemplarysystems exist that one skilled in the art could utilize (e.g., ReticLysate IVT Kit, Life Technologies, Waltham, MA).

An expression vector capable of expressing a polypeptide can also beprepared. Expression vectors for different cell types are well known inthe art and can be selected without undue experimentation. Generally,the DNA is inserted into an expression vector, such as a plasmid, inproper orientation and correct reading frame for expression. Ifnecessary, the DNA may be linked to the appropriate transcriptional andtranslational regulatory control nucleotide sequences recognized by thedesired host (e.g., bacteria), although such controls are generallyavailable in the expression vector. The vector is then introduced intothe host bacteria for cloning using standard techniques (see, e.g.,Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.).

Expression vectors comprising the isolated polynucleotides, as well ashost cells containing the expression vectors, are also contemplated. Theneoantigenic peptides may be provided in the form of RNA or cDNAmolecules encoding the desired neoantigenic peptides. One or moreneoantigenic peptides of the invention may be encoded by a singleexpression vector.

The term “polynucleotide encoding a polypeptide” encompasses apolynucleotide which includes only coding sequences for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequences. Polynucleotides can be in the form of RNA or inthe form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; andcan be double-stranded or single-stranded, and if single stranded can bethe coding strand or non-coding (anti-sense) strand.

In embodiments, the polynucleotides may comprise the coding sequence forthe tumor specific neoantigenic peptide fused in the same reading frameto a polynucleotide which aids, for example, in expression and/orsecretion of a polypeptide from a host cell (e.g., a leader sequencewhich functions as a secretory sequence for controlling transport of apolypeptide from the cell). The polypeptide having a leader sequence isa preprotein and can have the leader sequence cleaved by the host cellto form the mature form of the polypeptide.

In embodiments, the polynucleotides can comprise the coding sequence forthe tumor specific neoantigenic peptide fused in the same reading frameto a marker sequence that allows, for example, for purification of theencoded polypeptide, which may then be incorporated into thepersonalized neoplasia vaccine or immunogenic composition. For example,the marker sequence can be a hexa-histidine tag supplied by a pQE-9vector to provide for purification of the mature polypeptide fused tothe marker in the case of a bacterial host, or the marker sequence canbe a hemagglutinin (HA) tag derived from the influenza hemagglutininprotein when a mammalian host (e.g., COS-7 cells) is used. Additionaltags include, but are not limited to, Calmodulin tags, FLAG tags, Myctags, S tags, SBP tags, Softag 1, Softag 3, V5 tag, Xpress tag,Isopeptag, SpyTag, Biotin Carboxyl Carrier Protein (BCCP) tags, GSTtags, fluorescent protein tags (e.g., green fluorescent protein tags),maltose binding protein tags, Nus tags, Strep-tag, thioredoxin tag, TCtag, Ty tag, and the like.

In embodiments, the polynucleotides may comprise the coding sequence forone or more of the tumor specific neoantigenic peptides fused in thesame reading frame to create a single concatamerized neoantigenicpeptide construct capable of producing multiple neoantigenic peptides.

In certain embodiments, isolated nucleic acid molecules having anucleotide sequence at least 60% identical, at least 65% identical, atleast 70% identical, at least 75% identical, at least 80% identical, atleast 85% identical, at least 90% identical, at least 95% identical, orat least 96%, 97%, 98% or 99% identical to a polynucleotide encoding atumor specific neoantigenic peptide of the present invention, can beprovided.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence is intended that thenucleotide sequence of the polynucleotide is identical to the referencesequence except that the polynucleotide sequence can include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence. In other words, to obtain a polynucleotide having a nucleotidesequence at least 95% identical to a reference nucleotide sequence, upto 5% of the nucleotides in the reference sequence can be deleted orsubstituted with another nucleotide, or a number of nucleotides up to 5%of the total nucleotides in the reference sequence can be inserted intothe reference sequence. These mutations of the reference sequence canoccur at the amino- or carboxy-terminal positions of the referencenucleotide sequence or anywhere between those terminal positions,interspersed either individually among nucleotides in the referencesequence or in one or more contiguous groups within the referencesequence.

As a practical matter, whether any particular nucleic acid molecule isat least 80% identical, at least 85% identical, at least 90% identical,and in some embodiments, at least 95%, 96%, 97%, 98%, or 99% identicalto a reference sequence can be determined conventionally using knowncomputer programs such as the Bestfit program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, WI 53711). Bestfituses the local homology algorithm of Smith and Waterman, Advances inApplied Mathematics 2:482-489 (1981), to find the best segment ofhomology between two sequences. When using Bestfit or any other sequencealignment program to determine whether a particular sequence is, forinstance, 95% identical to a reference sequence according to the presentinvention, the parameters are set such that the percentage of identityis calculated over the full length of the reference nucleotide sequenceand that gaps in homology of up to 5% of the total number of nucleotidesin the reference sequence are allowed.

The isolated tumor specific neoantigenic peptides described herein canbe produced in vitro (e.g., in the laboratory) by any suitable methodknown in the art. Such methods range from direct protein syntheticmethods to constructing a DNA sequence encoding isolated polypeptidesequences and expressing those sequences in a suitable transformed host.In some embodiments, a DNA sequence is constructed using recombinanttechnology by isolating or synthesizing a DNA sequence encoding awild-type protein of interest. Optionally, the sequence can bemutagenized by site-specific mutagenesis to provide functional analogsthereof. See, e.g. Zoeller et al., Proc. Nat′l. Acad. Sci. USA81:5662-5066 (1984) and U.S. Pat. No. 4,588,585.

In embodiments, a DNA sequence encoding a polypeptide of interest wouldbe constructed by chemical synthesis using an oligonucleotidesynthesizer. Such oligonucleotides can be designed based on the aminoacid sequence of the desired polypeptide and selecting those codons thatare favored in the host cell in which the recombinant polypeptide ofinterest is produced. Standard methods can be applied to synthesize anisolated polynucleotide sequence encoding an isolated polypeptide ofinterest. For example, a complete amino acid sequence can be used toconstruct a back-translated gene. Further, a DNA oligomer containing anucleotide sequence coding for the particular isolated polypeptide canbe synthesized. For example, several small oligonucleotides coding forportions of the desired polypeptide can be synthesized and then ligated.The individual oligonucleotides typically contain 5′ or 3′ overhangs forcomplementary assembly.

Once assembled (e.g., by synthesis, site-directed mutagenesis, oranother method), the polynucleotide sequences encoding a particularisolated polypeptide of interest is inserted into an expression vectorand optionally operatively linked to an expression control sequenceappropriate for expression of the protein in a desired host. Properassembly can be confirmed by nucleotide sequencing, restriction mapping,and expression of a biologically active polypeptide in a suitable host.As well known in the art, in order to obtain high expression levels of atransfected gene in a host, the gene can be operatively linked totranscriptional and translational expression control sequences that arefunctional in the chosen expression host.

Recombinant expression vectors may be used to amplify and express DNAencoding the tumor specific neoantigenic peptides. Recombinantexpression vectors are replicable DNA constructs which have synthetic orcDNA-derived DNA fragments encoding a tumor specific ncoantigenicpeptide or a bioequivalent analog operatively linked to suitabletranscriptional or translational regulatory elements derived frommammalian, microbial, viral or insect genes. A transcriptional unitgenerally comprises an assembly of (1) a genetic element or elementshaving a regulatory role in gene expression, for example,transcriptional promoters or enhancers, (2) a structural or codingsequence which is transcribed into mRNA and translated into protein, and(3) appropriate transcription and translation initiation and terminationsequences, as described in detail herein. Such regulatory elements caninclude an operator sequence to control transcription. The ability toreplicate in a host, usually conferred by an origin of replication, anda selection gene to facilitate recognition of transformants canadditionally be incorporated. DNA regions are operatively linked whenthey are functionally related to each other. For example, DNA for asignal peptide (secretory leader) is operatively linked to DNA for apolypeptide if it is expressed as a precursor which participates in thesecretion of the polypeptide; a promoter is operatively linked to acoding sequence if it controls the transcription of the sequence; or aribosome binding site is operatively linked to a coding sequence if itis positioned so as to permit translation. Generally, operatively linkedmeans contiguous, and in the case of secretory leaders, means contiguousand in reading frame. Structural elements intended for use in yeastexpression systems include a leader sequence enabling extracellularsecretion of translated protein by a host cell. Alternatively, whererecombinant protein is expressed without a leader or transport sequence,it can include an N-terminal methionine residue. This residue canoptionally be subsequently cleaved from the expressed recombinantprotein to provide a final product.

Useful expression vectors for eukaryotic hosts, especially mammals orhumans include, for example, vectors comprising expression controlsequences from SV40, bovine papilloma virus, adenovirus andcytomegalovirus. Useful expression vectors for bacterial hosts includeknown bacterial plasmids, such as plasmids from Escherichia coli,including pCR 1, pBR322, pMB9 and their derivatives, wider host rangeplasmids, such as M13 and filamentous single-stranded DNA phages.

Suitable host cells for expression of a polypeptide include prokaryotes,yeast, insect or higher eukaryotic cells under the control ofappropriate promoters. Prokaryotes include gram negative or grampositive organisms, for example E. coli or bacilli. Higher eukaryoticcells include established cell lines of mammalian origin. Cell-freetranslation systems could also be employed. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are well known in the art (see Pouwels et al., CloningVectors: A Laboratory Manual, Elsevier, N.Y., 1985).

Various mammalian or insect cell culture systems are also advantageouslyemployed to express recombinant protein. Expression of recombinantproteins in mammalian cells can be performed because such proteins aregenerally correctly folded, appropriately modified and completelyfunctional. Examples of suitable mammalian host cell lines include theCOS-7 lines of monkey kidney cells, described by Gluzman (Cell 23:175,1981), and other cell lines capable of expressing an appropriate vectorincluding, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO),293, HeLa and BHK cell lines. Mammalian expression vectors can comprisenontranscribed elements such as an origin of replication, a suitablepromoter and enhancer linked to the gene to be expressed, and other 5′or 3′ flanking nontranscribed sequences, and 5′ or 3′ nontranslatedsequences, such as necessary ribosome binding sites, a polyadenylationsite, splice donor and acceptor sites, and transcriptional terminationsequences. Baculovirus systems for production of heterologous proteinsin insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47(1988).

The proteins produced by a transformed host can be purified according toany suitable method. Such standard methods include chromatography (e.g.,ion exchange, affinity and sizing column chromatography, and the like),centrifugation, differential solubility, or by any other standardtechnique for protein purification. Affinity tags such as hexahistidine,maltose binding domain, influenza coat sequence,glutathione-S-transferase, and the like can be attached to the proteinto allow easy purification by passage over an appropriate affinitycolumn. Isolated proteins can also be physically characterized usingsuch techniques as proteolysis, nuclear magnetic resonance and x-raycrystallography.

For example, supernatants from systems which secrete recombinant proteininto culture media can be first concentrated using a commerciallyavailable protein concentration filter, for example, an Amicon orMillipore Pellicon ultrafiltration unit. Following the concentrationstep, the concentrate can be applied to a suitable purification matrix.Alternatively, an anion exchange resin can be employed, for example, amatrix or substrate having pendant diethylaminoethyl (DEAE) groups. Thematrices can be acrylamide, agarose, dextran, cellulose or other typescommonly employed in protein purification. Alternatively, a cationexchange step can be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify a cancer stem cell protein-Fc composition.Some or all of the foregoing purification steps, in variouscombinations, can also be employed to provide a homogeneous recombinantprotein.

Recombinant protein produced in bacterial culture can be isolated, forexample, by initial extraction from cell pellets, followed by one ormore concentration, salting-out, aqueous ion exchange or size exclusionchromatography steps. High performance liquid chromatography (HPLC) canbe employed for final purification steps. Microbial cells employed inexpression of a recombinant protein can be disrupted by any convenientmethod, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

In Vivo Peptide/Polypeptide Synthesis

The present invention also contemplates the use of nucleic acidmolecules as vehicles for delivering neoantigenic peptides/polypeptidesto the subject in need thereof, in vivo, in the form of, e.g., DNA/RNAvaccines (see, e.g., WO2012/159643, and WO2012/159754, herebyincorporated by reference in their entirety).

In one embodiment neoantigens may be administered to a patient in needthereof by use of a plasmid. These are plasmids which usually consist ofa strong viral promoter to drive the in vivo transcription andtranslation of the gene (or complementary DNA) of interest (Mor, et al.,(1995). The Journal of Immunology 155 (4): 2039-2046). Intron A maysometimes be included to improve mRNA stability and hence increaseprotein expression (Leitner et al. (1997).The Journal of Immunology 159(12): 6112-6119). Plasmids also include a strongpolyadenylation/transcriptional termination signal, such as bovinegrowth hormone or rabbit beta-globulin polyadenylation sequences(Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42:343-410; Robinson et al., (2000). Adv. Virus Res. Advances in VirusResearch 55: 1-74; Böhmet al., (1996). Journal of Immunological Methods193 (1): 29-40.). Multicistronic vectors are sometimes constructed toexpress more than one immunogen, or to express an immunogen and animmunostimulatory protein (Lewis et al., (1999). Advances in VirusResearch (Academic Press) 54: 129-88).

Because the plasmid is the “vehicle” from which the immunogen isexpressed, optimising vector design for maximal protein expression isessential (Lewis et al., (1999). Advances in Virus Research (AcademicPress) 54: 129-88). One way of enhancing protein expression is byoptimising the codon usage of pathogenic mRNAs for eukaryotic cells.Another consideration is the choice of promoter. Such promoters may bethe SV40 promoter or Rous Sarcoma Virus (RSV).

Plasmids may be introduced into animal tissues by a number of differentmethods. The two most popular approaches are injection of DNA in saline,using a standard hypodermic needle, and gene gun delivery. A schematicoutline of the construction of a DNA vaccine plasmid and its subsequentdelivery by these two methods into a host is illustrated at ScientificAmerican (Weiner et al., (1999) Scientific American 281 (1): 34-41).Injection in saline is normally conducted intramuscularly (IM) inskeletal muscle, or intradermally (ID), with DNA being delivered to theextracellular spaces. This can be assisted by electroporation bytemporarily damaging muscle fibres with myotoxins such as bupivacainc;or by using hypertonic solutions of saline or sucrose (Alarcon et al.,(1999). Adv. Parasitol. Advances in Parasitology 42: 343-410). Immuneresponses to this method of delivery can be affected by many factors,including needle type, needle alignment, speed of injection, volume ofinjection, muscle type, and age, sex and physiological condition of theanimal being injected(Alarcon et al., (1999). Adv. Parasitol. Advancesin Parasitology 42: 343-410).

Gene gun delivery, the other commonly used method of delivery,ballistically accelerates plasmid DNA (pDNA) that has been adsorbed ontogold or tungsten microparticles into the target cells, using compressedhelium as an accelerant (Alarcon et al., (1999). Adv. Parasitol.Advances in Parasitology 42: 343-410; Lewis et al., (1999). Advances inVirus Research (Academic Press) 54: 129-88).

Alternative delivery methods may include aerosol instillation of nakedDNA on mucosal surfaces, such as the nasal and lung mucosa, (Lewis etal., (1999). Advances in Virus Research (Academic Press) 54: 129-88) andtopical administration of pDNA to the eye and vaginal mucosa (Lewis etal., (1999) Advances in Virus Research (Academic Press) 54: 129-88).Mucosal surface delivery has also been achieved using cationicliposomc-DNA preparations, biodegradable microspheres, attenuatedShigella or Listeria vectors for oral administration to the intestinalmucosa, and recombinant adenovirus vectors.

The method of delivery determines the dose of DNA required to raise aneffective immune response. Saline injections require variable amounts ofDNA, from 10 µg-1 mg, whereas gene gun deliveries require 100 to 1000times less DNA than intramuscular saline injection to raise an effectiveimmune response. Generally, 0.2 µg - 20 µg are required, althoughquantities as low as 16 ng have been reported. These quantities varyfrom species to species, with mice, for example, requiring approximately10 times less DNA than primates. Saline injections require more DNAbecause the DNA is delivered to the extracellular spaces of the targettissue (normally muscle), where it has to overcome physical barriers(such as the basal lamina and large amounts of connective tissue, tomention a few) before it is taken up by the cells, while gene gundeliveries bombard DNA directly into the cells, resulting in less“wastage” (See e.g., Sedegah et al., (1994). Proceedings of the NationalAcademy of Sciences of the United States of America 91 (21): 9866-9870;Daheshiaet al., (1997). The Journal of Immunology 159 (4): 1945-1952;Chen et al., (1998). The Journal of Immunology 160 (5): 2425-2432;Sizemore (1995) Science 270 (5234): 299-302; Fynan et al., (1993) Proc.Natl. Acad. Sci. U.S.A. 90 (24): 11479-82).

In one embodiment, a neoplasia vaccine or immunogenic composition mayinclude separate DNA plasmids encoding, for example, one or moreneoantigenic peptides/polypeptides as identified in according to theinvention. As discussed herein, the exact choice of expression vectorscan depend upon the peptide/polypeptides to be expressed, and is wellwithin the skill of the ordinary artisan. The expected persistence ofthe DNA constructs (e.g., in an episomal, non-replicating,non-integrated form in the muscle cells) is expected to provide anincreased duration of protection.

One or more neoantigenic peptides of the invention may be encoded andexpressed in vivo using a viral based system (e.g., an adenovirussystem, an adeno associated virus (AAV) vector, a poxvirus, or alentivirus). In one embodiment, the neoplasia vaccine or immunogeniccomposition may include a viral based vector for use in a human patientin need thereof, such as, for example, an adenovirus (see, e.g., Badenet al. First-in-human evaluation of the safety and immunogenicity of arecombinant adenovirus serotype 26 HIV-1 Env vaccine (IPCAVD 001). JInfect Dis. 2013 Jan 15;207(2):240-7, hereby incorporated by referencein its entirety). Plasmids that can be used for adeno associated virus,adenovirus, and lentivirus delivery have been described previously (seee.g., U.S. Pat. Nos. 6,955,808 and 6,943,019, and U.S. Pat. applicationNo. 20080254008, hereby incorporated by reference).

Among vectors that may be used in the practice of the invention,integration in the host genome of a cell is possible with retrovirusgene transfer methods, often resulting in long term expression of theinserted transgene. In a preferred embodiment the retrovirus is alentivirus. Additionally, high transduction efficiencies have beenobserved in many different cell types and target tissues. The tropism ofa retrovirus can be altered by incorporating foreign envelope proteins,expanding the potential target population of target cells. A retroviruscan also be engineered to allow for conditional expression of theinserted transgene, such that only certain cell types are infected bythe lentivirus. Cell type specific promoters can be used to targetexpression in specific cell types. Lentiviral vectors are retroviralvectors (and hence both lentiviral and retroviral vectors may be used inthe practice of the invention). Moreover, lentiviral vectors arepreferred as they are able to transduce or infect non-dividing cells andtypically produce high viral titers. Selection of a retroviral genetransfer system may therefore depend on the target tissue. Retroviralvectors are comprised of cis-acting long terminal repeats with packagingcapacity for up to 6-10 kb of foreign sequence. The minimum cis-actingLTRs are sufficient for replication and packaging of the vectors, whichare then used to integrate the desired nucleic acid into the target cellto provide permanent expression. Widely used retroviral vectors that maybe used in the practice of the invention include those based upon murineleukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immunodeficiency virus (SIV), human immuno deficiency virus (HIV), andcombinations thereof (see, e.g., Buchscher et al., (1992) J. Virol.66:2731-2739; Johann et al., (1992) J. Virol. 66:1635-1640; Sommnerfeltet al., (1990) Virol. 176:58-59; Wilson et al., (1998) J. Virol.63:2374-2378; Miller et al., (1991) J. Virol. 65:2220-2224;PCT/US94/05700). Zou et al. administered about 10 µl of a recombinantlentivirus having a titer of 1 x 10⁹ transducing units (TU)/ml by anintrathecal catheter. These sort of dosages can be adapted orextrapolated to use of a retroviral or lentiviral vector in the presentinvention.

Also useful in the practice of the invention is a minimal non-primatelentiviral vector, such as a lentiviral vector based on the equineinfectious anemia virus (EIAV) (see, e.g., Balagaan, (2006) J Gene Med;8: 275 - 285, Published online 21 Nov. 2005 in Wiley InterScience(www.interscience.wiley.com). DOI: 10.1002/jgm.845). The vectors mayhave cytomegalovirus (CMV) promoter driving expression of the targetgene. Accordingly, the invention contemplates amongst vector(s) usefulin the practice of the invention: viral vectors, including retroviralvectors and lentiviral vectors.

Also useful in the practice of the invention is an adenovirus vector.One advantage is the ability of recombinant adenoviruses to efficientlytransfer and express recombinant genes in a variety of mammalian cellsand tissues in vitro and in vivo, resulting in the high expression ofthe transferred nucleic acids. Further, the ability to productivelyinfect quiescent cells, expands the utility of recombinant adenoviralvectors. In addition, high expression levels ensure that the products ofthe nucleic acids will be expressed to sufficient levels to generate animmune response (see e.g., U.S. Pat. No. 7,029,848, hereby incorporatedby reference).

In an embodiment herein the delivery is via an adenovirus, which may beat a single booster dose containing at least 1 × 10⁵ particles (alsoreferred to as particle units, pu) of adenoviral vector. In anembodiment herein, the dose preferably is at least about 1 × 10⁶particles (for example, about 1 × 10⁶-1 x 10¹² particles), morepreferably at least about 1 × 10⁷ particles, more preferably at leastabout 1 x 10⁸ particles (e.g., about 1 × 10⁸-1 × 10¹¹ particles or about1 × 10⁸-1 × 10¹² particles), and most preferably at least about 1 × 10⁹particles (e.g., about 1 × 10⁹-1× 10¹⁰ particles or about 1 × 10⁹-1 ×10¹² particles), or even at least about 1 × 10¹⁰ particles (e.g., about1 × 10¹⁰-1 × 10¹² particles) of the adenoviral vector. Alternatively,the dose comprises no more than about 1 × 10¹⁴ particles, preferably nomore than about 1 × 10¹³ particles, even more preferably no more thanabout 1 × 10¹² particles, even more preferably no more than about 1 ×10¹¹ particles, and most preferably no more than about 1 × 10¹⁰particles (e.g., no more than about 1 × 10⁹ articles). Thus, the dosemay contain a single dose of adenoviral vector with, for example, about1 × 10⁶ particle units (pu), about 2 × 10⁶ pu, about 4 × 10⁶ pu, about 1× 10⁷ pu, about 2 × 10⁷ pu, about 4 × 10⁷ pu, about 1 × 10⁸ pu, about 2× 10⁸ pu, about 4 × 10⁸ pu, about 1 × 10⁹ pu, about 2 × 10⁹ pu, about 4× 10⁹ pu, about 1 × 10¹⁰ pu, about 2 × 10¹⁰ pu, about 4 × 10¹⁰ pu, about1 × 10¹¹ pu, about 2 × 10¹¹ pu, about 4 × 10¹¹ pu, about 1 × 10¹² pu,about 2 × 10¹² pu, or about 4 × 10¹² pu of adenoviral vector. See, forexample, the adenoviral vectors in U.S. Pat. No. 8,454,972 B2 to Nabel,et. al., granted on Jun. 4, 2013; incorporated by reference herein, andthe dosages at col 29, lines 36-58 thereof. In an embodiment herein, theadenovirus is delivered via multiple doses.

In terms of in vivo delivery, AAV is advantageous over other viralvectors due to low toxicity and low probability of causing insertionalmutagenesis because it doesn’t integrate into the host genome. AAV has apackaging limit of 4.5 or 4.75 Kb. Constructs larger than 4.5 or 4.75 Kbresult in significantly reduced virus production. There are manypromoters that can be used to drive nucleic acid molecule expression.AAV ITR can serve as a promoter and is advantageous for eliminating theneed for an additional promoter element. For ubiquitous expression, thefollowing promoters can be used: CMV, CAG, CBh, PGK, SV40, Ferritinheavy or light chains, etc. For brain expression, the followingpromoters can be used: Synapsinl for all neurons, CaMKIIalpha forexcitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc.Promoters used to drive RNA synthesis can include: Pol III promoterssuch as U6 or H1. The use of a Pol II promoter and intronic cassettescan be used to express guide RNA (gRNA).

As to AAV, the AAV can be AAV1, AAV2, AAV5 or any combination thereof.One can select the AAV with regard to the cells to be targeted; e.g.,one can select AAV serotypes 1, 2, 5 or a hybrid capsid AAV1, AAV2, AAV5or any combination thereof for targeting brain or neuronal cells; andone can select AAV4 for targeting cardiac tissue. AAV8 is useful fordelivery to the liver. The above promoters and vectors are preferredindividually.

In an embodiment herein, the delivery is via an AAV. A therapeuticallyeffective dosage for in vivo delivery of the AAV to a human is believedto be in the range of from about 20 to about 50 ml of saline solutioncontaining from about 1 × 10¹⁰ to about 1 × 10⁵⁰ functional AAV/mlsolution. The dosage may be adjusted to balance the therapeutic benefitagainst any side effects. In an embodiment herein, the AAV dose isgenerally in the range of concentrations of from about 1 × 10⁵ to 1 ×10⁵⁰ genomes AAV, from about 1 × 10⁸ to 1 × 10²⁰ genomes AAV, from about1 × 10¹⁰ to about 1 × 10¹⁶ genomes, or about 1 × 10¹¹ to about 1 × 10¹⁶genomes AAV. A human dosage may be about 1 × 10¹¹ genomes AAV. Suchconcentrations may be delivered in from about 0.001 ml to about 100 ml,about 0.05 to about 50 ml, or about 10 to about 25 ml of a carriersolution. In a preferred embodiment, AAV is used with a titer of about 2× 10¹³ viral genomes/milliliter, and each of the striatal hemispheres ofa mouse receives one 500 nanoliter injection. Other effective dosagescan be readily established by one of ordinary skill in the art throughroutine trials establishing dose response curves. See, for example, U.S.Pat. No. 8,404,658 B2 to Hajjar, et al., granted on Mar. 26, 2013, atcol. 27, lines 45-60.

In another embodiment effectively activating a cellular immune responsefor a neoplasia vaccine or immunogenic composition can be achieved byexpressing the relevant neoantigens in a vaccine or immunogeniccomposition in a non-pathogenic microorganism. Well-known examples ofsuch microorganisms are Mycobacterium bovis BCG, Salmonella andPseudomona (See, U.S. Pat. No. 6,991,797, hereby incorporated byreference in its entirety).

In another embodiment a Poxvirus is used in the neoplasia vaccine orimmunogenic composition. These include orthopoxvirus, avipox, vaccinia,MVA, NYVAC, canarypox, ALVAC, fowlpox, TROVAC, etc. (see e.g., Verardietal., Hum Vaccin Immunother. 2012 Jul;8(7):961-70; and Moss, Vaccine.2013; 31(39): 4220-4222). Poxvirus expression vectors were described in1982 and quickly became widely used for vaccine development as well asresearch in numerous fields. Advantages of the vectors include simpleconstruction, ability to accommodate large amounts of foreign DNA andhigh expression levels.

In another embodiment the vaccinia virus is used in the neoplasiavaccine or immunogenic composition to express a neoantigen. (Rolph etal., Recombinant viruses as vaccines and immunological tools. Curr OpinImmunol 9:5 17-524, 1997). The recombinant vaccinia virus is able toreplicate within the cytoplasm of the infected host cell and thepolypeptide of interest can therefore induce an immune response.Moreover, Poxviruses have been widely used as vaccine or immunogeniccomposition vectors because of their ability to target encoded antigensfor processing by the major histocompatibility complex class I pathwayby directly infecting immune cells, in particular antigen-presentingcells, but also due to their ability to self-adjuvant.

In another embodiment ALVAC is used as a vector in a neoplasia vaccineor immunogenic composition. ALVAC is a canarypox virus that can bemodified to express foreign transgenes and has been used as a method forvaccination against both prokaryotic and eukaryotic antigens (Horig H,Lee DS, Conkright W, et al. Phase I clinical trial of a recombinantcanarypoxvirus (ALVAC) vaccine expressing human carcinoembryonic antigenand the B7.1 co-stimulatory molecule. Cancer Immunol Immunother2000;49:504-14, von Mehren M, Arlen P, Tsang KY, et al. Pilot study of adual gene recombinant avipox vaccine containing both carcinoembryonicantigen (CEA) and B7.1 transgenes in patients with recurrentCEA-expressing adenocarcinomas. Clin Cancer Res 2000;6:22 19---28; MuseyL, Ding Y, Elizaga M, et al. HIV-1 vaccination administeredintramuscularly can induce both systemic and mucosal T cell immunity inHIV-1-uninfected individuals. J Immunol 2003;171:1094-101; Paoletti E.Applications of pox virus vectors to vaccination: an update. Proc NatlAcad Sci USA 1996;93:11349-53; U.S. Pat. No. 7,255,862). In a phase Iclinical trial, an ALVAC virus expressing the tumor antigen CEA showedan excellent safety profile and resulted in increased CEA-specificT-cell responses in selected patients; objective clinical responses,however, were not observed (Marshall JL, Hawkins MJ, Tsang KY, et al.Phase I study in cancer patients of a replication-defective avipoxrecombinant vaccine that expresses human carcinoembryonic antigen. JClin Oncol 1999;17:332-7).

In another embodiment a Modified Vaccinia Ankara (MVA) virus may be usedas a viral vector for a neoantigen vaccine or immunogenic composition.MVA is a member of the Orthopoxvirus family and has been generated byabout 570 serial passages on chicken embryo fibroblasts of the Ankarastrain of Vaccinia virus (CVA) (for review see Mayr, A., et al.,Infection 3, 6-14, 1975). As a consequence of these passages, theresulting MVA virus contains 31 kilobases less genomic informationcompared to CVA, and is highly host-cell restricted (Meyer, H. et al.,J. Gen. Virol. 72, 1031-1038, 1991). MVA is characterized by its extremeattenuation, namely, by a diminished virulence or infectious ability,but still holds an excellent immunogenicity. When tested in a variety ofanimal models, MVA was proven to be avirulent, even in immuno-suppressedindividuals. Moreover, MVA-BNⓇ-HER2 is a candidate immunotherapydesigned for the treatment of HER-2-positive breast cancer and iscurrently in clinical trials. (Mandl et al., Cancer Immunol Immunother.January 2012; 61(1): 19-29). Methods to make and use recombinant MVA hasbeen described (e.g., see U.S. Pat. Nos. 8,309,098 and 5,185,146 herebyincorporated in its entirety).

In another embodiment the modified Copenhagen strain of vaccinia virus,NYVAC and NYVAC variations are used as a vector (see U.S. Pat. No.7,255,862; PCT WO 95/30018; U.S. Pat. Nos. 5,364,773 and 5,494,807,hereby incorporated by reference in its entirety).

In one embodiment recombinant viral particles of the vaccine orimmunogenic composition are administered to patients in need thereof.Dosages of expressed neoantigen can range from a few to a few hundredmicrograms, e.g., 5 to 500 .mu.g. The vaccine or immunogenic compositioncan be administered in any suitable amount to achieve expression atthese dosage levels. The viral particles can be administered to apatient in need thereof or transfected into cells in an amount of aboutat least 10^(3.5) pfu; thus, the viral particles are preferablyadministered to a patient in need thereof or infected or transfectedinto cells in at least about 10⁴ pfu to about 10⁶ pfu; however, apatient in need thereof can be administered at least about 10⁸ pfu suchthat a more preferred amount for administration can be at least about10⁷ pfu to about 10⁹ pfu. Doses as to NYVAC are applicable as to ALVAC,MVA, MVA-BN, and avipoxes, such as canarypox and fowlpox.

Vaccine or Immunogenic Composition Adjuvant

Effective vaccine or immunogenic compositions advantageously include astrong adjuvant to initiate an immune response. As described herein,poly-lCLC, an agonist of TLR3 and the RNA helicase -domains of MDA5 andRIG3, has shown several desirable properties for a vaccine orimmunogenic composition adjuvant. These properties include the inductionof local and systemic activation of immune cells in vivo, production ofstimulatory chemokines and cytokines, and stimulation ofantigen-presentation by DCs. Furthermore, poly-ICLC can induce durableCD4+ and CD8+ responses in humans. Importantly, striking similarities inthe upregulation of transcriptional and signal transduction pathwayswere seen in subjects vaccinated with poly-ICLC and in volunteers whohad received the highly effective, replication-competent yellow fevervaccine. Furthermore, >90% of ovarian carcinoma patients immunized withpoly-lCLC in combination with a NY -ESO-I peptide vaccine (in additionto Montanide) showed induction of CD4+ and CD8+ T cell, as well asantibody responses to the peptide in a recent phase 1 study. At the sametime, poly-ICLC has been extensively tested in more than 25 clinicaltrials to date and exhibited a relatively benign toxicity profile. Inaddition to a powerful and specific immunogen the neoantigen peptidesmay be combined with an adjuvant (e.g., poly-ICLC) or anotheranti-neoplastic agent. Without being bound by theory, these neoantigensare expected to bypass central thymic tolerance (thus allowing strongeranti-tumor T cell response), while reducing the potential forautoimmunity (e.g., by avoiding targeting of normal self-antigens). Aneffective immune response advantageously includes a strong adjuvant toactivate the immune system (Speiser and Romero, Molecularly definedvaccines for cancer immunotherapy, and protective T cell immunitySeminars in Immunol 22:144 (2010)). For example, Toll-like receptors(TLRs) have emerged as powerful sensors of microbial and viral pathogen“danger signals”, effectively inducing the innate immune system, and inturn, the adaptive immune system (Bhardwaj and Gnjatic, TLR. AGONISTS:Are They Good Adjuvants? Cancer J. 16:382-391 (2010)). Among the TLRagonists, poly-ICLC (a synthetic doublestranded RNA mimic) is one of themost potent activators of myeloid-derived dendritic cells. In a humanvolunteer study, poly-ICLC has been shown to be safe and to induce agene expression profile in peripheral blood cells comparable to thatinduced by one of the most potent live attenuated viral vaccines, theyellow fever vaccine YF-17D (Caskey et al, Synthetic doublestranded RNAinduces innate immune responses similar to a live viral vaccine inhumans J Exp Med 208:2357 (2011)). In a preferred embodiment Hiltonol®,a GMP preparation of poly-ICLC prepared by Oncovir, Inc, is utilized asthe adjuvant. In other embodiments, other adjuvants described herein areenvisioned. For instance oil-in-water, water-in-oil or multiphasicW/O/W, see, e.g., US 7,608,279 and Aucouturier et al, Vaccine 19 (2001),2666-2672, and documents cited therein.

Checkpoint Inhibitors

The present invention features methods of treating or preventing aneoplasia comprising the steps of administering to a subject a neoplasiavaccine or immunogenic composition, as described herein, and at leastone checkpoint inhibitor. Accordingly, 1, 2, 3, 4, 5, or more checkpointinhibitors may be administered. In certain exemplary embodiments, onecheckpoint inhibitor is administered. In other exemplary embodiments, 2checkpoint inhibitors arc administered.

Page et al. (Annu. Rev. Med. 2014.65) summarizes published trialsinvestigating checkpoint modulators in solid tumors. Mullard, A. (NatureReviews, Drug Discovery. Vol. 12, July 2013) provides a review ofcheckpoint inhibitors. A summary table of exemplary checkpointinhibitors is provided herein.

Drug Lead company Most advanced indications Phase Anti-PD1 NivulumabBristol-Myers Squibb Renal cell cancer, melanoma, NSCLC IIILambrolizumab Merck & Co. Melanoma II Pidillzumab* Curetech Colorectalcancer, melanoma. DLBCL II AMP-224 GlaxoSmithKline Solid tumours IAnti-PDL1 MEDI-4736 AstraZeneca Solid tumours I MPDL3280A RocheMelanoma, solid tumours I

Anti-CTLA4 Antibodies

Cytotoxic T-lymphocyte-associated antigen (CTLA-4), also known as CD152,is a co-inhibitory molecule that functions to regulate T-cellactivation.

CTLA4 was initially identified as negative regulator on the surface ofT-cells that was upregulated shortly after initiation of a de novoimmune response or stimulation of an existing response in order todampen the subsequent immune T-cell response and prevent auto-immunityor uncontrolled inflammation. Thus, the magnitude of the developingimmune response has been closely tied to CTLA4 action. In certainembodiments, the anti-CTLA4 antibody is Ipilumumab or Tremelimumab.

Checkpoint inhibitors function by modulating the immune system’sendogenous mechanisms of T cell regulation. Ipilimumab (YERVOY,Bristol-Meyers Squibb, New York, NY)- is a monoclonal antibody and isthe first such checkpoint inhibitor to be approved by the US Food andDrug Administration (FDA)- has become standard treatment for metastaticmelanoma (Hodi et al., N. Engl. J. Med. 363:711-23. 2010; Robert et al.,N. Engl. J. Med. 364:2517-26. 2011). Ipilimumab binds and blocksinhibitory signaling mediated by the T cell surface co-inhibitorymolecule cytotoxic T lymphocyte antigen 4 (CTLA-4). Because themechanism of action is not specific to one tumor type, and because awealth of preclinical data supports the role of tumor immunesurveillance across multiple malignancies (Andre et al., Clin. CancerRes. 19:28-33. 2013; May et al. Clin. Cancer Res.17:5233-38. 2011),Ipilumumab is being investigated as a treatment for patients withprostate, lung, renal, and breast cancer, among other tumor types.Ipilimumab works by activating the immune system by targeting CTLA-4.

Another CTLA-4-blocking antibody, Tremelimumab, continues to beinvestigated in clinical trials and has also demonstrated durableresponses in patients with melanoma (Kirkwood et al., Clin. Cancer Res.16:1042-48. 2010; Ribas et al. J. Clin. Oncol. 31:616-22, 2013).

Accordingly, the present invention features in exemplary embodiments,novel combinations of a neoplasia vaccine or immunogenic composition andone or more anti-CTLA4 antibodies. The invention also features in otherexemplary embodiments, novel combinations of a neoplasia vaccine orimmunogenic composition, Ipilimumab and/or Nivolumab and one or moreanti-CTLA4 antibodies.

Inhibitors of Programmed Cell Death-1 Pathway

Whereas CTLA-4 serves to regulate early T cell activation, ProgrammedDeath-1 (PD-1) signaling functions in part to regulate T cell activationin peripheral tissues. The PD-1 receptor refers to an immunoinhibitoryreceptor belonging to the CD28 family. PD-1 is expressed on a number ofcell types including T regs, activated B cells, and natural killer (NK)cells, and is expressed predominantly on previously activated T cells invivo, and binds to two ligands, PD-L1 and PD-L2. PD1′s endogenousligands, PD-L1 and PD-L2, are expressed in activated immune cells aswell as nonhematopoietic cells, including tumor cells. PD-1 as usedherein is meant to include human PD-1 (hPD-1), variants, isoforms, andspecies homologs of hPD-1, and analogs having at least one commonepitope with hPD-1. The complete hPD-l sequence can be found underGENBANK Accession No. U64863. Programmed Death Ligand-1 (PD-L1″ is oneof two cell surface glycoprotein ligands for PD-1 (the other beingPD-L2) that downregulate T cell activation and cytokine secretion uponbinding to PD-1. PD-L1 as used herein includes human PD-L1 (hPD)-L1),variants, isoforms, and species homologs of hPD-L1, and analogs havingat least one common epitope with hPD-L1. The complete hPD-L1 sequencecan be found under GENBANK Accession No. Q9NZQ7. Tumors have beendemonstrated to escape immune surveillance by expressing PD-L1/L2,thereby suppressing tumor-infiltrating lymphocytes via PD-1/PD-L1,2interactions (Dong et al. Nat. Med. 8:793-800. 2002). Inhibition ofthese interactions with therapeutic antibodies has been shown to enhanceT cell response and stimulate antitumor activity (Freeman et al. J. Exp.Med. 192: 1027-34.2000).

The Abs of the invention include, but are not limited to, all of theanti-PD-1 and anti-PD-L1 Abs disclosed in U.S. Pat. Nos. 8,008,449 and7,943,743, respectively. Other anti-PD-1 mAbs have been described in,for example, U.S. Pat. Nos. 7,488,802 and 8,168,757, and anti-PD-L1 mAbshave been described in, for example, U.S. Pat. Nos. 7,635,757 and8,217,149, and U.S. Publication No. 2009/0317368. U.S. Pat. No.8,008,449 exemplifies seven anti-PD-1 HuMAbs: 17D8, 2D3, 4H1. 5C4 (alsoreferred to herein as nivolumab or BMS-936558), 4A11, 7D3 and 5F4.

In some embodiments, the anti-PD-1 antibody is nivolumab. Alternativenames for Nivolumab include MDX- 1 106, MDX-1 106-04, ONO-4538,BMS-936558. In some embodiments, the anti-PD- 1 antibody is Nivolumab(CAS Registry Number: 946414-94-4).

Nivolumab) is a fully human lgG4 blocking monoclonal antibody againstPD-1 (Topaliam et al., N. Engl. J. Med. 366:2443-54. 2012). Nivolumabspecifically blocks PD-1, which can overcome immune resistance. Theligands for PD-1 have been identified as PD-L1 (B7-H1), which isexpressed on all hemopoietic cells and many nonhemopoietic tissues, andPD-L2 (B7-DC), whose expression is restricted primarily to dendriticcells and macrophages (Dong, H. et al. 1999. Nat. Med. 5:1365; Freeman,G. J.et al. 2000. J. Exp. Med. 192:1027; Latchman, Y. et al. 2001. Nat.Immunol. 2:261; Tseng, S. Y. et al. 2001. J. Exp. Med. 193:839). PD-L1is overexpressed in many cancers and is often associated with poorprognosis (Okazaki T et al., Intern. Immun. 2007 19(7):813) (Thompson RHet al., Cancer Res 2006, 66(7):3381). Interestingly, the majority oftumor infiltrating T lymphocytes predominantly express PD-1 , incontrast to T lymphocytes in normal tissues and peripheral blood Tlymphocytes indicating that up-regulation of PD- I on tumor-reactive Tcells can contribute to impaired antitumor immune responses (Blood 20091 14(8): 1537). Specifically, since tumor cells express PD-L1, animmunosuppressive PD-1 ligand, inhibition of the interaction betweenPD-1 and PD-L1 can enhance T-cell responses in vitro and mediatepreclinical antitumor activity.

A number of clinical trials (Phase I, II and III) involving Nivolumabhave been conducted or are on-going (seeclinicaltrials.gov/ct2/results?term=nivolumab&pg=1, accessed on Dec. 20,2013). For example, in a phase I dose escalation trial, nivolumab wassafe, and objective responses were 16-31% across tumor types, with mostresponses being durable for >1 year (Topaliam et al., Presented at Annu.Meet. Am. Soc. Clin. Oncol., Chicago, May 31-June 4. 2013). In anotherstudy, the safety and clinical activity of nivolumab (anti-PD-1,BMS-936558, ONO-4538) in combination with ipilimumab in patients withadvanced melanoma was investigated (Wolchok, J Clin Oncol 31, 2013(suppl; abstr 9012 2013 AS(:O Annual Meeting).

Two anti-PD-L1 inhibitory antibodies, MPDL3280A (Genentech, South SanFrancisco, CA) and BMS-936559 (Bristol Meyers Squibb, New York, NY),have undergone clinical investigation. Like nivolumab and MK-3475, theseantibodies are thought to function principally by blocking PD-1/PD-I_ tsignaling. Unlike PD-1 antibodies, PD-L1 antibodies spare potentialinteractions between PD-L2 and PD-1, but additionally block interactionsbetween PD-L1 and CD80 (Park et al., 2010. Blood 116:1291-98). MPDL3280Ahas been evaluated in multiple tumor types, with safety and preliminaryefficacy identified in melanoma; renal cell carcinoma; non-small celllung carcinoma (NSCLC); and colorectal, gastric, and head/neck squamouscell carcinoma (Herbst et al. resented at Annu. Meet. Am. Soc. Clin.Oncol., Chicago. May 31-June 4. 2013 ). Similarly, BMS-936559 was shownto be safe and clinically active across multiple tumor types in a phaseI trial. MEDI-4736 is another PD-L1-blocking antibody currently inclinical development (NCT0 1693562).

In addition to CTLA-4 and PD-1/PD-L1, numerous other immunomodulatorytargets have been identified preclinically, many with correspondingtherapeutic antibodies that are being investigated in clinical trials.Page et al. (Annu. Rev. Med. 2014.65) details targets of antibody immunemodulators in FIG. 1 , incorporated by reference herein.

The present invention features in exemplary aspects, novel combinationsof a neoplasia vaccine or immunogenic composition and one or moreinhibitors of the PD-1 pathway. In preferred embodiments, the inhibitorof the PD-1 pathway is an anti-PD1 antibody, for example Nivolumab.

The present invention also features in other exemplary aspects, novelcombinations of a neoplasia vaccine or immunogenic composition andNivolumab and/or one or more anti-CTLA4 antibodies.

Indications

Examples of cancers and cancer conditions that can be treated with thecombination therapy of this document include, but are not limited to apatient in need thereof that has been diagnosed as having cancer, or atrisk of developing cancer. The subject may have a solid tumor such asbreast, ovarian, prostate, lung, kidney, gastric, colon, testicular,head and neck, pancreas, brain, melanoma, and other tumors of tissueorgans and hematological tumors, such as lymphomas and leukemias,including acute myelogenous leukemia, chronic myelogenous leukemia,chronic lymphocytic leukemia, T cell lymphocytic leukemia, and B celllymphomas, tumors of the brain and central nervous system (e.g., tumorsof the meninges, brain, spinal cord, cranial nerves and other parts ofthe CNS, such as glioblastomas or medulla blastomas); head and/or neckcancer, breast tumors, tumors of the circulatory system (e.g., heart,mediastinum and pleura, and other intrathoracic organs, vascular tumors,and tumor-associated vascular tissue); tumors of the blood and lymphaticsystem (e.g., Hodgkin’s disease, Non-Hodgkin’s disease lymphoma,Burkitt’s lymphoma, AIDS-related lymphomas, malignantimmunoproliferative diseases, multiple myeloma, and malignant plasmacell neoplasms, lymphoid leukemia, myeloid leukemia, acute or chroniclymphocytic leukemia, monocytic leukemia, other leukemias of specificcell type, leukemia of unspecified cell type, unspecified malignantneoplasms of lymphoid, hematopoietic and related tissues, such asdiffuse large cell lymphoma, T-cell lymphoma or cutaneous T-celllymphoma); tumors of the excretory system (e.g., kidney, renal pelvis,ureter, bladder, and other urinary organs); tumors of thegastrointestinal tract (e.g., esophagus, stomach, small intestine,colon, colorectal, rectosigmoid junction, rectum, anus, and anal canal);tumors involving the liver and intrahepatic bile ducts, gall bladder,and other parts of the biliary tract, pancreas, and other digestiveorgans; tumors of the oral cavity (e.g., lip, tongue, gum, floor ofmouth, palate, parotid gland, salivary glands, tonsil, oropharynx,nasopharynx, puriform sinus, hypopharynx, and other sites of the oralcavity); tumors of the reproductive system (e.g., vulva, vagina, Cervixuteri, uterus, ovary, and other sites associated with female genitalorgans, placenta, penis, prostate, testis, and other sites associatedwith male genital organs); tumors of the respiratory tract (e.g., nasalcavity, middle car, accessory sinuses, larynx, trachea, bronchus andlung, such as small cell lung cancer and non-small cell lung cancer);tumors of the skeletal system (e.g., bone and articular cartilage oflimbs, bone articular cartilage and other sites); tumors of the skin(e.g., malignant melanoma of the skin, non-melanoma skin cancer, basalcell carcinoma of skin, squamous cell carcinoma of skin, mesothelioma,Kaposi’s sarcoma); and tumors involving other tissues includingperipheral nerves and autonomic nervous system, connective and softtissue, retroperitoneoum and peritoneum, eye, thyroid, adrenal gland,and other endocrine glands and related structures, secondary andunspecified malignant neoplasms of lymph nodes, secondary malignantneoplasm of respiratory and digestive systems and secondary malignantneoplasm of other sites.

Of special interest is the treatment of Non-Hodgkin’s Lymphoma (NHL),clear cell Renal Cell Carcinoma (ccRCC), metastatic melanoma, sarcoma,leukemia or a cancer of the bladder, colon, brain, breast, head andneck, endometrium, lung, ovary, pancreas or prostate. In certainembodiments, the melanoma is high risk melanoma.

Cancers that can be treated using this combination therapy may includeamong others cases which are refractory to treatment with otherchemotherapeutics. The term “refractory, as used herein refers to acancer (and/or metastases thereof), which shows no or only weakantiproliferative response (e.g., no or only weak inhibition of tumorgrowth) after treatment with another chemotherapeutic agent. These arecancers that cannot be treated satisfactorily with otherchemotherapeutics. Refractory cancers encompass not only (i) cancerswhere one or more chemotherapeutics have already failed during treatmentof a patient, but also (ii) cancers that can be shown to be refractoryby other means, e.g., biopsy and culture in the presence ofchemotherapeutics.

The combination therapy described herein is also applicable to thetreatment of patients in need thereof who have not been previouslytreated.

The combination therapy described herein is also applicable where thesubject has no detectable neoplasia but is at high risk for diseaserecurrence.

Also of special interest is the treatment of patients in need thereof swho have undergone Autologous Hematopoietic Stem Cell Transplant(AHSCT), and in particular patients who demonstrate residual diseaseafter undergoing AHSCT. The post-AHSCT setting is characterized by a lowvolume of residual disease, the infusion of immune cells to a situationof homeostatic expansion, and the absence of any standardrelapse-delaying therapy. These features provide a unique opportunity touse the claimed neoplastic vaccine or immunogenic composition andcheckpoint inhibitor compositions to delay disease relapse.

Pharmaceutical Compositions/Methods of Delivery

The present invention is also directed to pharmaceutical compositionscomprising an effective amount of one or more compounds according to thepresent invention (including a pharmaceutically acceptable salt,thereof), optionally in combination with a pharmaceutically acceptablecarrier, excipient or additive.

When administered as a combination, the therapeutic agents (i.e. theneoplasia vaccine or immunogenic composition and one or more checkpointinhibitors) can be formulated as separate compositions that are given atthe same time or different times, or the therapeutic agents can be givenas a single composition.

The compositions may be administered once daily, twice daily, once everytwo days, once every three days, once every four days, once every fivedays, once every six days, once every seven days, once every two weeks,once every three weeks, once every four weeks, once every two months,once every six months, or once per year. The dosing interval can beadjusted according to the needs of individual patients. For longerintervals of administration, extended release or depot formulations canbe used.

The compositions of the invention can be used to treat diseases anddisease conditions that are acute, and may also be used for treatment ofchronic conditions. In particular, the compositions of the invention areused in methods to treat or prevent a neoplasia.

In certain embodiments, the compounds of the invention are administeredfor time periods exceeding two weeks, three weeks, one month, twomonths, three months, four months, five months, six months, one year,two years, three years, four years, or five years, ten years, or fifteenyears; or for example, any time period range in days, months or years inwhich the low end of the range is any time period between 14 days and 15years and the upper end of the range is between 15 days and 20 years(e.g., 4 weeks and 15 years, 6 months and 20 years). In some cases, itmay be advantageous for the compounds of the invention to beadministered for the remainder of the patient’s life. In preferredembodiments, the patient is monitored to check the progression of thedisease or disorder, and the dose is adjusted accordingly. In preferredembodiments, treatment according to the invention is effective for atleast two weeks, three weeks, one month, two months, three months, fourmonths, five months, six months, one year, two years, three years, fouryears, or five years, ten years, fifteen years, twenty years, or for theremainder of the subject’s life.

As described herein, in certain embodiments, administration of thecheckpoint inhibitor is initiated before initiation of administration ofthe neoplasia vaccine or immunogenic composition. In other embodiments,administration of the checkpoint inhibitor is initiated after initiationof administration of the neoplasia vaccine or immunogenic composition.In still other embodiments, administration of the checkpoint inhibitoris initiated simultaneously with the initiation of administration of theneoplasia vaccine or immunogenic composition.

Administration of the checkpoint inhibitor may continue every 2, 3, 4,5, 6, 7, 8 or more weeks after the first administration of thecheckpoint inhibitor. It is understood that week 1 is meant to includedays 1-7, week 2 is meant to include days 8-14, week 3 is meant toinclude days 15-21 and week 4 is meant to include days 22-28. Whendosing is described as being on weekly intervals it means approximately7 days apart although in any given week the day can be one or more daysbefore or after the scheduled day

In certain embodiments, administration of the checkpoint inhibitor iswithheld during the week prior to administration of the neoplasiavaccine or immunogenic composition. In other embodiments, administrationof the checkpoint inhibitor is withheld during administration of theneoplasia vaccine or immunogenic composition.

Surgical resection uses surgery to remove abnormal tissue in cancer,such as mediastinal, neurogenic, or germ cell tumors, or thymoma. Incertain embodiments, administration of the checkpoint inhibitor isinitiated following tumor resection. In other embodiments,administration of the neoplasia vaccine or immunogenic composition isinitiated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or moreweeks after tumor resection. Preferably, administration of the neoplasiavaccine or immunogenic composition is initiated 4, 5, 6, 7, 8, 9, 10, 11or 12 weeks after tumor resection.

Prime/ boost regimens refer to the successive administrations of avaccine or immunogenic or immunological compositions. In certainembodiments, administration of the neoplasia vaccine or immunogeniccomposition is in a prime/ boost dosing regimen, for exampleadministration of the neoplasia vaccine or immunogenic composition atweeks 1, 2, 3 or 4 as a prime and administration of the neoplasiavaccine or immunogenic composition is at months 2, 3 or 4 as a boost. Inanother embodiment heterologous prime-boost strategies are used toellicit a greater cytotoxic T-cell response (see Schneider et al.,Induction of CD8+ T cells using heterologous prime-boost immunisationstrategies, Immunological Reviews Volume 170, Issue 1, pages 29-38,August 1999). In another embodiment DNA encoding neoantigens is used toprime followed by a protein boost. In another embodiment protein is usedto prime followed by boosting with a virus encoding the neoantigen. Inanother embodiment a virus encoding the neoantigen is used to prime andanother virus is used to boost. In another embodiment protein is used toprime and DNA is used to boost. In a preferred embodiment a DNA vaccineor immunogenic composition is used to prime a T-cell response and arecombinant viral vaccine or immunogenic composition is used to boostthe response. In another preferred embodiment a viral vaccine orimmunogenic composition is coadministered with a protein or DNA vaccineor immunogenic composition to act as an adjuvant for the protein or DNAvaccine or immunogenic composition. The patient can then be boosted witheither the viral vaccine or immunogenic composition, protein, or DNAvaccine or immunogenic composition (see Hutchings et al., Combination ofprotein and viral vaccines induces potent cellular and humoral immuneresponses and enhanced protection from murine malaria challenge. InfectImmun. 2007 Dec;75(12):5819-26. Epub 2007 Oct 1).

The pharmaceutical compositions can be processed in accordance withconventional methods of pharmacy to produce medicinal agents foradministration to patients in need thereof, including humans and othermammals.

Modifications of the neoantigenic peptides can affect the solubility,bioavailability and rate of metabolism of the peptides, thus providingcontrol over the delivery of the active species. Solubility can beassessed by preparing the neoantigenic peptide and testing according toknown methods well within the routine practitioner’s skill in the art.

It has been found that a pharmaceutical composition comprising succinicacid or a pharmaceutically acceptable salt thereof (succinate) canprovide improved solubility for the neoantigenic peptides. Thus, in oneaspect, the invention provides a pharmaceutical composition comprising:at least one neoantigenic peptide or a pharmaceutically acceptable saltthereof; a pH modifier (such as a base, such as a dicarboxylate ortricarboxylate salt, for example, a pharmaceutically acceptable salt ofsuccinic acid or citric acid); and a pharmaceutically acceptablecarrier. Such pharmaceutical compositions can be prepared by combining asolution comprising at least one neoantigenic peptide with a base, suchas a dicarboxylate or tricarboxylate salt, such as a pharmaceuticallyacceptable salt of succinic acid or citric acid (such as sodiumsuccinate), or by combining a solution comprising at least oneneoantigenic peptide with a solution comprising a base, such as adicarboxylate or tricarboxylate salt, such as a pharmaceuticallyacceptable salt of succinic acid or citric acid (including, e.g., asuccinate buffer solution). In certain embodiments, the pharmaceuticalcomposition comprises sodium succinate. In certain embodiments, the pHmodifier (such as citrate or succinate) is present in the composition ata concentration from about 1 mM to about 10 mM, and, in certainembodiments, at a concentration from about 1.5 mM to about 7.5 mM, orabout 2.0 to about 6.0 mM, or about 3.75 to about 5.0 mM.

In certain embodiments of the pharmaceutical composition thepharmaceutically acceptable carrier comprises water. In certainembodiments, the pharmaceutically acceptable carrier further comprisesdextrose. In certain embodiments, the pharmaceutically acceptablecarrier further comprises dimethylsulfoxide. In certain embodiments, thepharmaceutical composition further comprises an immunomodulator oradjuvant. In certain embodiments, the immunodulator or adjuvant isselected from the group consisting of poly-ICLC, 1018 ISS, aluminumsalts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF.IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX,JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide TMS 1312,Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174,OM-197-MP-EC, ONTAK, PEPTEL, vector system, PLGA microparticles,resiquimod, SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, and Aquila’s QS21 stimulon. Incertain embodiments, the immunomodulator or adjuvant comprisespoly-ICLC.

Xanthenone derivatives such as, for example, Vadimezan or AsA404 (alsoknown as 5,6-dimethylaxanthenone-4-acetic acid (DMXAA)), may also beused as adjuvants according to embodiments of the invention.Alternatively, such derivatives may also be administered in parallel tothe vaccine or immunogenic composition of the invention, for example viasystemic or intratumoral delivery, to stimulate immunity at the tumorsite. Without being bound by theory, it is believed that such xanthenonederivatives act by stimulating interferon (IFN) production via thestimulator of IFN gene ISTING) receptor (see e.g., Conlon et al. (2013)Mouse, but not Human STING, Binds and Signals in Response to theVascular Disrupting Agent 5,6-Dimethylxanthenone-4-Acetic Acid, Journalof Immunology, 190:5216-25 and Kim et al. (2013) Anticancer Flavonoidsare Mouse-Selective STING Agonists, 8:1396-1401).

The vaccine or immunological composition may also include an adjuvantcompound chosen from the acrylic or methacrylic polymers and thecopolymers of maleic anhydride and an alkenyl derivative. It is inparticular a polymer of acrylic or methacrylic acid cross-linked with apolyalkenyl ether of a sugar or polyalcohol (carbomer), in particularcross-linked with an allyl sucrose or with allylpentaerythritol. It mayalso be a copolymer of maleic anhydride and ethylene cross-linked, forexample, with divinyl ether (see U.S. Pat. No. 6,713,068 herebyincorporated by reference in its entirety)..

In certain embodiments, the pH modifier can stabilize the adjuvant orimmunomodulator as described herein.

In certain embodiments, a pharmaceutical composition comprises: one tofive peptides, dimethylsulfoxide (DMSO), dextrose, water, succinate,poly I: poly C, poly-L-lysine, carboxymethylcellulose, and chloride. Incertain embodiments, each of the one to five peptides is present at aconcentration of 300 µg/ml. In certain embodiments, the pharmaceuticalcomposition comprises ≤ 3% DMSO by volume. In certain embodiments, thepharmaceutical composition comprises 3.6 .... 3.7 % dextrose in water.In certain embodiments, the pharmaceutical composition comprises 3.6 -3.7 mM succinate (e.g., as sodium succinate). In certain embodiments,the pharmaceutical composition comprises 0.5 mg/ml poly I: poly C. Incertain embodiments, the pharmaceutical composition comprises 0.375mg/ml poly-L-Lysine. In certain embodiments, the pharmaceuticalcomposition comprises 1.25 mg/ml sodium carboxymethylcellulose. Incertain embodiments, the pharmaceutical composition comprises 0.225%sodium chloride.

Pharmaceutical compositions comprise the herein-described tumor specificneoantigenic peptides in a therapeutically effective amount for treatingdiseases and conditions (e.g., a neoplasia/tumor), which have beendescribed herein, optionally in combination with a pharmaceuticallyacceptable additive, carrier and/or excipient. One of ordinary skill inthe art from this disclosure and the knowledge in the art will recognizethat a therapeutically effective amount of one of more compoundsaccording to the present invention may vary with the condition to betreated, its severity, the treatment regimen to be employed, thepharmacokinetics of the agent used, as well as the patient (animal orhuman) treated.

To prepare the pharmaceutical compositions according to the presentinvention, a therapeutically effective amount of one or more of thecompounds according to the present invention is preferably intimatelyadmixed with a pharmaceutically acceptable carrier according toconventional pharmaceutical compounding techniques to produce a dose. Acarrier may take a wide variety of forms depending on the form ofpreparation desired for administration, e.g., ocular, oral, topical orparenteral, including gels, creams ointments, lotions and time releasedimplantable preparations, among numerous others. In preparingpharmaceutical compositions in oral dosage form, any of the usualpharmaceutical media may be used. Thus, for liquid oral preparationssuch as suspensions, elixirs and solutions, suitable carriers andadditives including water, glycols, oils, alcohols, flavoring agents,preservatives, coloring agents and the like may be used. For solid oralpreparations such as powders, tablets, capsules, and for solidpreparations such as suppositories, suitable carriers and additivesincluding starches, sugar carriers, such as dextrose, mannitol, lactoseand related carriers, diluents, granulating agents, lubricants, binders,disintegrating agents and the like may be used. If desired, the tabletsor capsules may be enteric-coated or sustained release by standardtechniques.

The active compound is included in the pharmaceutically acceptablecarrier or diluent in an amount sufficient to deliver to a patient atherapeutically effective amount for the desired indication, withoutcausing serious toxic effects in the patient treated.

Oral compositions generally include an inert diluent or an ediblecarrier. They may be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound or its prodrug derivative can be incorporated with excipientsand used in the form of tablets, troches, or capsules. Pharmaceuticallycompatible binding agents, and/or adjuvant materials can be included aspart of the composition.

The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a bindersuch as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose, a dispersing agent such as alginicacid or corn starch; a lubricant such as magnesium stearate; a glidantsuch as colloidal silicon dioxide; a sweetening agent such as sucrose orsaccharin; or a flavoring agent such as peppermint, methyl salicylate,or orange flavoring. When the dosage unit form is a capsule, it cancontain, in addition to material herein discussed, a liquid carrier suchas a fatty oil. In addition, dosage unit forms can contain various othermaterials which modify the physical form of the dosage unit, forexample, coatings of sugar, shellac, or enteric agents.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous liquidor a non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil emulsion and as a bolus, etc.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, preservative, surface-active ordispersing agent. Molded tablets may be made by molding in a suitablemachine a mixture of the powdered compound moistened with an inertliquid diluent. The tablets optionally may be coated or scored and maybe formulated so as to provide slow or controlled release of the activeingredient therein.

Methods of formulating such slow or controlled release compositions ofpharmaceutically active ingredients, are known in the art and describedin several issued U.S. Patents, some of which include, but are notlimited to, U.S. Pat. Nos. 3,870,790; 4,226,859; 4,369,172; 4,842,866and 5,705,190, the disclosures of which are incorporated herein byreference in their entireties. Coatings can be used for delivery ofcompounds to the intestine (see, e.g., U.S. Pat. Nos. 6,638,534,5,541,171, 5,217,720, and 6,569,457, and references cited therein).

The active compound or pharmaceutically acceptable salt thereof may alsobe administered as a component of an elixir, suspension, syrup, wafer,chewing gum or the like. A syrup may contain, in addition to the activecompounds, sucrose or fructose as a sweetening agent and certainpreservatives, dyes and colorings and flavors.

Solutions or suspensions used for ocular, parenteral, intradermal,subcutaneous, or topical application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates; and agents for theadjustment of tonicity such as sodium chloride or dextrose.

In certain embodiments, the pharmaceutically acceptable carrier is anaqueous solvent, i.e., a solvent comprising water, optionally withadditional co-solvents. Exemplary pharmaceutically acceptable carriersinclude water, buffer solutions in water (such as phosphate-bufferedsaline (PBS), and 5% dextrose in water (D5W). In certain embodiments,the aqueous solvent further comprises dimethyl sulfoxide (DMSO), e.g.,in an amount of about 1-4%, or 1-3%. In certain embodiments, thepharmaceutically acceptable carrier is isotonic (i.e., has substantiallythe same osmotic pressure as a body fluid such as plasma).

In one embodiment, the active compounds are prepared with carriers thatprotect the compound against rapid elimination from the body, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, polylactic acid, and polylactic-co-glycolicacid (PLGA). Methods for preparation of such formulations are within theambit of the skilled artisan in view of this disclosure and theknowledge in the art.

A skilled artisan from this disclosure and the knowledge in the artrecognizes that in addition to tablets, other dosage forms can beformulated to provide slow or controlled release of the activeingredient. Such dosage forms include, but are not limited to, capsules,granulations and gel-caps.

Liposomal suspensions may also be pharmaceutically acceptable carriers.These may be prepared according to methods known to those skilled in theart. For example, liposomal formulations may be prepared by dissolvingappropriate lipid(s) in an inorganic solvent that is then evaporated,leaving behind a thin film of dried lipid on the surface of thecontainer. An aqueous solution of the active compound are thenintroduced into the container. The container is then swirled by hand tofree lipid material from the sides of the container and to disperselipid aggregates, thereby forming the liposomal suspension. Othermethods of preparation well known by those of ordinary skill may also beused in this aspect of the present invention.

The formulations may conveniently be presented in unit dosage form andmay be prepared by conventional pharmaceutical techniques. Suchtechniques include the step of bringing into association the activeingredient and the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations arc prepared by uniformly and intimatelybringing into association the active ingredient with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

Formulations and compositions suitable for topical administration in themouth include lozenges comprising the ingredients in a flavored basis,usually sucrose and acacia or tragacanth; pastilles comprising theactive ingredient in an inert basis such as gelatin and glycerin, orsucrose and acacia; and mouthwashes comprising the ingredient to beadministered in a suitable liquid carrier.

Formulations suitable for topical administration to the skin may bepresented as ointments, creams, gels and pastes comprising theingredient to be administered in a pharmaceutical acceptable carrier. Apreferred topical delivery system is a transdermal patch containing theingredient to be administered.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising, for example, cocoa butter or asalicylate.

Formulations suitable for nasal administration, wherein the carrier is asolid, include a coarse powder having a particle size, for example, inthe range of 20 to 500 microns which is administered in the manner inwhich snuff is administered, i.e., by rapid inhalation through the nasalpassage from a container of the powder held close up to the nose.Suitable formulations, wherein the carrier is a liquid, foradministration, as for example, a nasal spray or as nasal drops, includeaqueous or oily solutions of the active ingredient.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

The parenteral preparation can be enclosed in ampoules, disposablesyringes or multiple dose vials made of glass or plastic. Ifadministered intravenously, preferred carriers include, for example,physiological saline or phosphate buffered saline (PBS).

For parenteral formulations, the carrier usually comprises sterile wateror aqueous sodium chloride solution, though other ingredients includingthose which aid dispersion may be included. Of course, where sterilewater is to be used and maintained as sterile, the compositions andcarriers are also sterilized. Injectable suspensions may also beprepared, in which case appropriate liquid carriers, suspending agentsand the like may be employed.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain antioxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example, sealed ampules and vials, and may be stored ina freeze-dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example, water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Administration of the active compound may range from continuous(intravenous drip) to several oral administrations per day (for example,Q.I.D.) and may include oral, topical, eye or ocular, parenteral,intramuscular, intravenous, sub-cutaneous, transdermal (which mayinclude a penetration enhancement agent), buccal and suppositoryadministration, among other routes of administration, including throughan eye or ocular route.

The neoplasia vaccine or immunogenic composition and the at least onecheckpoint inhibitor, and any additional agents, may be administered byinjection, orally, parenterally, by inhalation spray, rectally,vaginally, or topically in dosage unit formulations containingconventional pharmaceutically acceptable carriers, adjuvants, andvehicles. The term parenteral as used herein includes, into a lymph nodeor nodes, subcutaneous, intravenous, intramuscular, intrasternal,infusion techniques, intraperitoneally, eye or ocular, intravitreal,intrabuccal, transdermal, intranasal, into the brain, includingintracranial and intradural, into the joints, including ankles, knees,hips, shoulders, elbows, wrists, directly into tumors, and the like, andin suppository form.

In certain embodiments, the vaccine or immunogenic composition or theone of more checkpoint inhibitors are administered intravenously orsubcutaneously.

Application of the subject therapeutics may be local, so as to beadministered at the site of interest. In certain embodiments, thecheckpoint inhibitor is administered subcutaneously near the site ofadministration of the neoplasia vaccine or immunogenic composition, forexample within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 cm of the site ofvaccine or immunogenic composition administration, and preferably within5 cm of the site of administration of the neoplasia vaccine orimmunogenic composition. It is to be understood by one skilled in theart administering the compositions that the concentration of thecheckpoint inhibitor administered to the subject may be changed based onthe location of administration. For example, if the checkpoint inhibitoris administered near the site of administration of the neoplasia vaccineor immunogenic composition, then the concentration of the checkpointinhibitor may be decreased.

Various techniques can be used for providing the subject compositions atthe site of interest, such as injection, use of catheters, trocars,projectiles, pluronic gel, stents, sustained drug release polymers orother device which provides for internal access. Where an organ ortissue is accessible because of removal from the patient, such organ ortissue may be bathed in a medium containing the subject compositions,the subject compositions may be painted onto the organ, or may beapplied in any convenient way.

The tumor specific neoantigenic peptides may be administered through adevice suitable for the controlled and sustained release of acomposition effective in obtaining a desired local or systemicphysiological or pharmacological effect. The method includes positioningthe sustained released drug delivery system at an area wherein releaseof the agent is desired and allowing the agent to pass through thedevice to the desired area of treatment.

The tumor specific neoantigenic peptides may be utilized in combinationwith at least one known other therapeutic agent, or a pharmaceuticallyacceptable salt of said agent. Examples of known therapeutic agentswhich can be used for combination therapy include, but are not limitedto, corticosteroids (e.g., cortisone, prednisone, dexamethasone),non-steroidal antiinflammatory drugs (NSAIDS) (e.g., ibuprofen,celccoxib, aspirin, indomethicin, naproxen), alkylating agents such asbusulfan, cis-platin, mitomycin C, and carboplatin; antimitotic agentssuch as colchicine, vinblastine, paclitaxel, and docetaxel; topo Iinhibitors such as camptothecin and topotecan; topo II inhibitors suchas doxorubicin and etoposide; and/or RNA/DNA antimetabolites such as5-azacytidine, 5-fluorouracil and methotrexate; DNA antimetabolites suchas 5-fluoro-2′-deoxy-uridine, ara-C, hydroxyurea and thioguanine;antibodies such as HERCEPTIN and RITUXAN.

It should be understood that in addition to the ingredients particularlymentioned herein, the formulations of the present invention may includeother agents conventional in the art having regard to the type offormulation in question, for example, those suitable for oraladministration may include flavoring agents.

Pharmaceutically acceptable salt forms may be the preferred chemicalform of compounds according to the present invention for inclusion inpharmaceutical compositions according to the present invention.

The present compounds or their derivatives, including prodrug forms ofthese agents, can be provided in the form of pharmaceutically acceptablesalts. As used herein, the term pharmaceutically acceptable salts orcomplexes refers to appropriate salts or complexes of the activecompounds according to the present invention which retain the desiredbiological activity of the parent compound and exhibit limitedtoxicological effects to normal cells. Nonlimiting examples of suchsalts are (a) acid addition salts formed with inorganic acids (forexample, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoricacid, nitric acid, and the like), and salts formed with organic acidssuch as acetic acid, oxalic acid, tartaric acid, succinic acid, malicacid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginicacid, and polyglutamic acid, among others; (b) base addition saltsformed with metal cations such as zinc, calcium, sodium, potassium, andthe like, among numerous others.

The compounds herein are commercially available or can be synthesized.As can be appreciated by the skilled artisan, further methods ofsynthesizing the compounds of the formulae herein is evident to those ofordinary skill in the art. Additionally, the various synthetic steps maybe performed in an alternate sequence or order to give the desiredcompounds. Synthetic chemistry transformations and protecting groupmethodologies (protection and deprotection) useful in synthesizing thecompounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, 2nd. Ed., Wiley-VCH Publishers (1999); T.W. Greene andP.G.M. Wuts, Protective Groups in Organic Synthesis, 3rd. Ed., JohnWiley and Sons (1999); L. Fieser and M. Fieser, Fieser and Fieser’sReagents for Organic Synthesis, John Wiley and Sons (1999); and L.Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, JohnWiley and Sons (1995), and subsequent editions thereof.

Dosage

When the agents described herein are administered as pharmaceuticals tohumans or animals, they can be given per se or as a pharmaceuticalcomposition containing active ingredient in combination with apharmaceutically acceptable carrier, excipient, or diluent.

Actual dosage levels and time course of administration of the activeingredients in the pharmaceutical compositions of the invention can bevaried so as to obtain an amount of the active ingredient which iseffective to achieve the desired therapeutic response for a particularpatient, composition, and mode of administration, without being toxic tothe patient. Generally, agents or pharmaceutical compositions of theinvention are administered in an amount sufficient to reduce oreliminate symptoms associated with viral infection and/or autoimmunedisease.

A preferred dose of an agent is the maximum that a patient can tolerateand not develop serious or unacceptable side effects.

Determination of an effective amount is well within the capability ofthose skilled in the art, especially in light of the detailed disclosureprovided herein. Generally, an efficacious or effective amount of anagent is determined by first administering a low dose of the agent(s)and then incrementally increasing the administered dose or dosages untila desired effect (e.g., reduce or eliminate symptoms associated withviral infection or autoimmune disease) is observed in the treatedsubject, with minimal or acceptable toxic side effects. Applicablemethods for determining an appropriate dose and dosing schedule foradministration of a pharmaceutical composition of the present inventionare described, for example, in Goodman and Gilman’s The PharmacologicalBasis of Therapeutics, Goodman et al., eds., 1 1th Edition, McGraw-Hill2005, and Remington: The Science and Practice of Pharmacy, 20th and 21stEditions, Gennaro and University of the Sciences in Philadelphia, Eds.,Lippencott Williams & Wilkins (2003 and 2005), each of which is herebyincorporated by reference.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, as herein discussed, or an appropriate fractionthereof, of the administered ingredient.

The dosage regimen for treating a disorder or a disease with the tumorspecific neoantigenic peptides of this invention and/or compositions ofthis invention is based on a variety of factors, including the type ofdisease, the age, weight, sex, medical condition of the patient, theseverity of the condition, the route of administration, and theparticular compound employed. Thus, the dosage regimen may vary widely,but can be determined routinely using standard methods.

The amounts and dosage regimens administered to a subject can depend ona number of factors, such as the mode of administration, the nature ofthe condition being treated, the body weight of the subject beingtreated and the judgment of the prescribing physician; all such factorsbeing within the ambit of the skilled artisan from this disclosure andthe knowledge in the art.

The amount of compound included within therapeutically activeformulations according to the present invention is an effective amountfor treating the disease or condition.

In general, a therapeutically effective amount of the present preferredcompound in dosage form usually ranges from slightly less than about0.025 mg/kg/day to about 2.5 g/kg/day, preferably about 0.1 mg/kg/day toabout 100 mg/kg/day of the patient or considerably more, depending uponthe compound used, the condition or infection treated and the route ofadministration, although exceptions to this dosage range may becontemplated by the present invention. In its most preferred form,compounds according to the present invention are administered in amountsranging from about 1 mg/kg/day to about 100 mg/kg/day. The dosage of thecompound can depend on the condition being treated, the particularcompound, and other clinical factors such as weight and condition of thepatient and the route of administration of the compound. It is to beunderstood that the present invention has application for both human andveterinary use.

According to certain exemplary embodiments, the vaccine or immunogeniccomposition is administered at a dose of about 10 µg- 1 mg perneoantigenic peptide. According to certain exemplary embodiments, thevaccine or immunogenic composition is administered at an average weeklydose level of about 10 µg- 2000 µg per neoantigenic peptide. Accordingto certain exemplary embodiments, the checkpoint inhibitor isadministered at a dose of about 0.1-10 mg/kg. According to certainexemplary embodiments, the anti-CTLA4 antibody is administered at a doseof about 1 mg/kg- 3 mg/kg. For example, in certain exemplaryembodiments, Nivolumab isgin dosing at the standard single agent dosinglevel of 3 mg/kg. When the one or more checkpoint inhibitors areadministered at the site of administration of the vaccine or immunogeniccomposition, the inhibitor is preferably administered at a dose of about0.1-1 mg per site of administration of the neoplasia vaccine orimmunogenic composition.

Prefered embodiments use the concentrations and timings used in clinicaltrials for checkpoint inhibitors, alone or in combination with aneoantigen vaccine or immunogenic composition. Topalian, et al. N Engl JMed 2012; 366:2443-2454 describes a phase 1 study that assessed thesafety, antitumor activity, and pharmacokinetics of BMS-936558, a fullyhuman IgG4-blocking monoclonal antibody directed against PD-1, inpatients in need thereof with selected advanced solid tumors. Theantibody was administered as an intravenous infusion every 2 weeks ofeach 8-week treatment cycle. Response was assessed after each treatmentcycle. Patients received treatment for up to 2 years (12 cycles).Patients with advanced melanoma, non-small-cell lung cancer, renal-cellcancer, castration-resistant prostate cancer, or colorectal cancer wereenrolled. Cohorts of three to six patients per dose level were enrolledsequentially at doses of 1.0, 3.0, or 10.0 mg per kilogram of bodyweight. Initially, five expansion cohorts of approximately 16 patientseach were enrolled at doses of 10.0 mg per kilogram for melanoma,non-small-cell lung cancer, renal-cell cancer, castration-resistantprostate cancer, and colorectal cancer. On the basis of initial signalsof activity, additional expansion cohorts of approximately 16 patientseach were enrolled for melanoma (at a dose of 1.0 or 3.0 mg perkilogram, followed by cohorts randomly assigned to 0.1, 0.3, or 1.0 mgper kilogram), lung cancer (patients with the squamous or nonsquamoussubtype, randomly assigned to a dose of 1.0, 3.0, or 10.0 mg perkilogram), and renal-cell cancer (at a dose of 1.0 mg per kilogram).

Wolchok,et al. N Engl J Med 2013; 369:122-133, describes a clinicaltrial using Nivolumab plus Ipilimumab in advanced melanoma. In the studypatients were administered intravenous doses of nivolumab and ipilimumabevery 3 weeks for 4 doses, followed by nivolumab alone every 3 weeks for4 doses. The combined treatment was subsequently administered every 12weeks for up to 8 doses. In a sequenced regimen, patients previouslytreated with ipilimumab received nivolumab every 2 weeks for up to 48doses. The maximum doses that were associated with an acceptable levelof adverse events were nivolumab at a dose of 1 mg per kilogram of bodyweight and ipilimumab at a dose of 3 mg per kilogram.

Wolchok et al., Clin. Cancer Res. 15, 7412; 2009 describes a phase IIclinical trial program with ipilimumab. Patients were treated withinduction therapy (ipilimumab 10 mg/kg every 3 wk ×4) followed bymaintenance therapy in eligible patients (ipilimumab 10 mg/kg every 12wk, beginning at week 24).

Hamid et al., N Engl J Med 2013; 369:134-144, describes safety and tumorresponses with Lambrolizumab (Anti-PD-1) in melanoma. Patients withadvanced melanoma were administered lambrolizumab intravenously at adose of 10 mg per kilogram of body weight every 2 or 3 weeks or 2 mg perkilogram every 3 weeks. Patients included both those who had receivedprior treatment with the immune checkpoint inhibitor ipilimumab andthose who had not.

Spigel et al., J Clin Oncol 31, 2013 (suppl; abstr 8008) describe aphase I trial for MPDL3280A, a human monoclonal Ab containing anengineered Fc-domain designed to optimize efficacy and safety, targetingPD-Ll. Patients with squamous or nonsquamous NSCLC received MPDL3280A byIV at doses between 1-20 mg/kg for up to 1 y.

The concentration of active compound in the drug composition will dependon absorption, distribution, inactivation, and excretion rates of thedrug as well as other factors known to those of skill in the art. It isto be noted that dosage values will also vary with the severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that the concentration ranges set forth herein areexemplary only and are not intended to limit the scope or practice ofthe claimed composition. The active ingredient may be administered atonce, or may be divided into a number of smaller doses to beadministered at varying intervals of time.

The invention provides for pharmaceutical compositions containing atleast one tumor specific neoantigen described herein. In embodiments,the pharmaceutical compositions contain a pharmaceutically acceptablecarrier, excipient, or diluent, which includes any pharmaceutical agentthat does not itself induce the production of an immune response harmfulto a subject receiving the composition, and which may be administeredwithout undue toxicity. As used herein, the term “pharmaceuticallyacceptable” means being approved by a regulatory agency of the Federalor a state government or listed in the U.S. Pharmacopia, EuropeanPharmacopia or other generally recognized pharmacopia for use inmammals, and more particularly in humans. These compositions can beuseful for treating and/or preventing viral infection and/or autoimmunedisease.

A thorough discussion of pharmaceutically acceptable carriers, diluents,and other excipients is presented in Remington’s Pharmaceutical Sciences(17th ed., Mack Publishing Company) and Remington: The Science andPractice of Pharmacy (21st ed., Lippincott Williams & Wilkins), whichare hereby incorporated by reference. The formulation of thepharmaceutical composition should suit the mode of administration. Inembodiments, the pharmaceutical composition is suitable foradministration to humans, and can be sterile, non-particulate and/ornon-pyrogenic.

Pharmaceutically acceptable carriers, excipients, or diluents include,but are not limited, to saline, buffered saline, dextrose, water,glycerol, ethanol, sterile isotonic aqueous buffer, and combinationsthereof.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives, and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include, but arenot limited to: (1) water soluble antioxidants, such as ascorbic acid,cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodiumsulfite and the like; (2) oil-soluble antioxidants, such as ascorbylpalmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene(BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3)metal chelating agents, such as citric acid, ethylenediamine tetraaceticacid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

In embodiments, the pharmaceutical composition is provided in a solidform, such as a lyophilized powder suitable for reconstitution, a liquidsolution, suspension, emulsion, tablet, pill, capsule, sustained releaseformulation, or powder.

In embodiments, the pharmaceutical composition is supplied in liquidform, for example, in a sealed container indicating the quantity andconcentration of the active ingredient in the pharmaceuticalcomposition. In related embodiments, the liquid form of thepharmaceutical composition is supplied in a hermetically sealedcontainer.

Methods for formulating the pharmaceutical compositions of the presentinvention are conventional and well known in the art (see Remington andRemington’s). One of skill in the art can readily formulate apharmaceutical composition having the desired characteristics (e.g.,route of administration, biosafety, and release profile).

Methods for preparing the pharmaceutical compositions include the stepof bringing into association the active ingredient with apharmaceutically acceptable carrier and, optionally, one or moreaccessory ingredients. The pharmaceutical compositions can be preparedby uniformly and intimately bringing into association the activeingredient with liquid carriers, or finely divided solid carriers, orboth, and then, if necessary, shaping the product. Additionalmethodology for preparing the pharmaceutical compositions, including thepreparation of multilayer dosage forms, are described in Ansel’sPharmaceutical Dosage Forms and Drug Delivery Systems (9th cd.,Lippincott Williams & Wilkins), which is hereby incorporated byreference.

Pharmaceutical compositions suitable for oral administration can be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound(s) describedherein, a derivative thereof, or a pharmaceutically acceptable salt orprodrug thereof as the active ingredient(s). The active ingredient canalso be administered as a bolus, electuary, or paste.

In solid dosage forms for oral administration (e.g., capsules, tablets,pills, dragees, powders, granules and the like), the active ingredientis mixed with one or more pharmaceutically acceptable carriers,excipients, or diluents, such as sodium citrate or dicalcium phosphate,and/or any of the following: (1) fillers or extenders, such as starches,lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders,such as, for example, carboxymethylcellulose, alginates, gelatin,polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such asglycerol; (4) disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and sodium carbonate; (5) solution retarding agents, such as paraffin;(6) absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, acetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets, and pills, thepharmaceutical compositions can also comprise buffering agents. Solidcompositions of a similar type can also be prepared using fillers insoft and hard-filled gelatin capsules, and excipients such as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

A tablet can be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets can be prepared usingbinders (for example, gelatin or hydroxypropylmethyl cellulose),lubricants, inert diluents, preservatives, disintegrants (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-actives, and/ or dispersing agents. Molded tablets can be madeby molding in a suitable machine a mixture of the powdered activeingredient moistened with an inert liquid diluent.

The tablets and other solid dosage forms, such as dragees, capsules,pills, and granules, can optionally be scored or prepared with coatingsand shells, such as enteric coatings and other coatings well known inthe art.

In some embodiments, in order to prolong the effect of an activeingredient, it is desirable to slow the absorption of the compound fromsubcutaneous or intramuscular injection. This can be accomplished by theuse of a liquid suspension of crystalline or amorphous material havingpoor water solubility. The rate of absorption of the active ingredientthen depends upon its rate of dissolution which, in turn, can dependupon crystal size and crystalline form. Alternatively, delayedabsorption of a parenterally-administered active ingredient isaccomplished by dissolving or suspending the compound in an oil vehicle.In addition, prolonged absorption of the injectable pharmaceutical formcan be brought about by the inclusion of agents that delay absorptionsuch as aluminum monostearate and gelatin.

Controlled release parenteral compositions can be in form of aqueoussuspensions, microspheres, microcapsules, magnetic microspheres, oilsolutions, oil suspensions, emulsions, or the active ingredient can beincorporated in biocompatible carrier(s), liposomes, nanoparticles,implants or infusion devices.

Materials for use in the preparation of microspheres and/ormicrocapsules include biodegradable/biocrodible polymers such aspolyglactin, poly-(isobutyl cyanoacrylate),poly(2-hydroxyethyl-L-glutamine) and poly(lactic acid).

Biocompatible carriers which can be used when formulating a controlledrelease parenteral formulation include carbohydrates such as dextrans,proteins such as albumin, lipoproteins or antibodies.

Materials for use in implants can be non-biodegradable, e.g.,polydimethylsiloxane, or biodegradable such as, e.g.,poly(caprolactone), poly(lactic acid), poly(glycolic acid) or poly(orthoesters).

In embodiments, the active ingredient(s) are administered by aerosol.This is accomplished by preparing an aqueous aerosol, liposomalpreparation, or solid particles containing the compound. A nonaqueous(e.g., fluorocarbon propellant) suspension can be used. Thepharmaceutical composition can also be administered using a sonicnebulizer, which would minimize exposing the agent to shear, which canresult in degradation of the compound.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the active ingredient(s) together withconventional pharmaceutically-acceptable carriers and stabilizers. Thecarriers and stabilizers vary with the requirements of the particularcompound, but typically include nonionic surfactants (Tweens, Pluronics,or polyethylene glycol), innocuous proteins like serum albumin, sorbitanesters, oleic acid, lecithin, amino acids such as glycine, buffers,salts, sugars or sugar alcohols. Aerosols generally are prepared fromisotonic solutions.

Dosage forms for topical or transdermal administration of an activeingredient(s) includes powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The activeingredient(s) can be mixed under sterile conditions with apharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants as appropriate.

Transdermal patches suitable for use in the present invention aredisclosed in Transdermal Drug Delivery: Developmental Issues andResearch Initiatives (Marcel Dekker Inc., 1989) and U.S. Pat. Nos.4,743,249, 4,906,169, 5,198,223, 4,816,540, 5,422,119, 5,023,084, whichare hereby incorporated by reference. The transdermal patch can also beany transdermal patch well known in the art, including transscrotalpatches. Pharmaceutical compositions in such transdermal patches cancontain one or more absorption enhancers or skin permeation enhancerswell known in the art (see, e.g., U.S. Pat. Nos. 4,379,454 and4,973,468, which are hereby incorporated by reference). Transdermaltherapeutic systems for use in the present invention can be based oniontophoresis, diffusion, or a combination of these two effects.

Transdermal patches have the added advantage of providing controlleddelivery of active ingredient(s) to the body. Such dosage forms can bemade by dissolving or dispersing the active ingredient(s) in a propermedium. Absorption enhancers can also be used to increase the flux ofthe active ingredient across the skin. The rate of such flux can becontrolled by either providing a rate controlling membrane or dispersingthe active ingredient(s) in a polymer matrix or gel.

Such pharmaceutical compositions can be in the form of creams,ointments, lotions, liniments, gels, hydrogels, solutions, suspensions,sticks, sprays, pastes, plasters and other kinds of transdermal drugdelivery systems. The compositions can also include pharmaceuticallyacceptable carriers or excipients such as emulsifying agents,antioxidants, buffering agents, preservatives, humectants, penetrationenhancers, chelating agents, gel-forming agents, ointment bases,perfumes, and skin protective agents.

Examples of emulsifying agents include, but are not limited to,naturally occurring gums, e.g. gum acacia or gum tragacanth, naturallyoccurring phosphatides, e.g. soybean lecithin and sorbitan monooleatederivatives.

Examples of antioxidants include, but are not limited to, butylatedhydroxy anisole (BHA), ascorbic acid and derivatives thereof, tocopheroland derivatives thereof, and cysteine.

Examples of preservatives include, but are not limited to, parabens,such as methyl or propyl p-hydroxybenzoate and benzalkonium chloride.

Examples of humectants include, but are not limited to, glycerin,propylene glycol, sorbitol and urea.

Examples of penetration enhancers include, but are not limited to,propylene glycol, DMSO, triethanolamine, N,N-dimethylacetamide,N,N-dimethylformamide, 2-pyrrolidone and derivatives thereof,tetrahydrofurfuryl alcohol, propylene glycol, diethylene glycolmonoethyl or monomethyl ether with propylene glycol monolaurate ormethyl laurate, eucalyptol, lecithin, TRANSCUTOL, and AZONE.

Examples of chelating agents include, but are not limited to, sodiumEDTA, citric acid and phosphoric acid.

Examples of gel forming agents include, but are not limited to,Carbopol, cellulose derivatives, bentonite, alginates, gelatin andpolyvinylpyrrolidone.

In addition to the active ingredient(s), the ointments, pastes, creams,and gels of the present invention can contain excipients, such as animaland vegetable fats, oils, waxes, paraffins, starch, tragacanth,cellulose derivatives, polyethylene glycols, silicones, bentonites,silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain excipients such as lactose, talc, silicicacid, aluminum hydroxide, calcium silicates and polyamide powder, ormixtures of these substances. Sprays can additionally contain customarypropellants, such as chlorofluorohydrocarbons, and volatileunsubstituted hydrocarbons, such as butane and propane.

Injectable depot forms are made by forming microencapsule matrices ofcompound(s) of the invention in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of compound topolymer, and the nature of the particular polymer employed, the rate ofcompound release can be controlled. Examples of other biodegradablepolymers include poly(orthoesters) and poly(anhydrides). Depotinjectable formulations are also prepared by entrapping the drug inliposomes or microemulsions which are compatible with body tissue.

Subcutaneous implants are well known in the art and are suitable for usein the present invention. Subcutaneous implantation methods arepreferably non-irritating and mechanically resilient. The implants canbe of matrix type, of reservoir type, or hybrids thereof. In matrix typedevices, the carrier material can be porous or non-porous, solid orsemi-solid, and permeable or impermeable to the active compound orcompounds. The carrier material can be biodegradable or may slowly erodeafter administration. In some instances, the matrix is nondegradable butinstead relies on the diffusion of the active compound through thematrix for the carrier material to degrade. Alternative subcutaneousimplant methods utilize reservoir devices where the active compound orcompounds are surrounded by a rate controlling membrane, e.g., amembrane independent of component concentration (possessing zero-orderkinetics). Devices consisting of a matrix surrounded by a ratecontrolling membrane also suitable for use.

Both reservoir and matrix type devices can contain materials such aspolydimethylsiloxane, such as SILASTIC, or other silicone rubbers.Matrix materials can be insoluble polypropylene, polyethylene, polyvinylchloride, ethylvinyl acetate, polystyrene and polymethacrylate, as wellas glycerol esters of the glycerol palmitostearate, glycerol stearate,and glycerol behenate type. Materials can be hydrophobic or hydrophilicpolymers and optionally contain solubilizing agents.

Subcutaneous implant devices can be slow-release capsules made with anysuitable polymer, e.g., as described in U.S. Pat. Nos. 5,035,891 and4,210,644, which are hereby incorporated by reference.

In general, at least four different approaches are applicable in orderto provide rate control over the release and transdermal permeation of adrug compound. These approaches are: membrane-moderated systems,adhesive diffusion-controlled systems, matrix dispersion-type systemsand microreservoir systems. It is appreciated that a controlled releasepercutaneous and/or topical composition can be obtained by using asuitable mixture of these approaches.

In a membrane-moderated system, the active ingredient is present in areservoir which is totally encapsulated in a shallow compartment moldedfrom a drug-impermeable laminate, such as a metallic plastic laminate,and a rate-controlling polymeric membrane such as a microporous or anon-porous polymeric membrane, e.g., ethylene-vinyl acetate copolymer.The active ingredient is released through the rate controlling polymericmembrane. In the drug reservoir, the active ingredient can either bedispersed in a solid polymer matrix or suspended in an unleachable,viscous liquid medium such as silicone fluid. On the external surface ofthe polymeric membrane, a thin layer of an adhesive polymer is appliedto achieve an intimate contact of the transdermal system with the skinsurface. The adhesive polymer is preferably a polymer which ishypoallergenic and compatible with the active drug substance.

In an adhesive diffusion-controlled system, a reservoir of the activeingredient is formed by directly dispersing the active ingredient in anadhesive polymer and then by, e.g., solvent casting, spreading theadhesive containing the active ingredient onto a flat sheet ofsubstantially drug-impermeable metallic plastic backing to form a thindrug reservoir layer.

A matrix dispersion-type system is characterized in that a reservoir ofthe active ingredient is formed by substantially homogeneouslydispersing the active ingredient in a hydrophilic or lipophilic polymermatrix. The drug-containing polymer is then molded into disc with asubstantially well-defined surface area and controlled thickness. Theadhesive polymer is spread along the circumference to form a strip ofadhesive around the disc.

A microrcscrvoir system can be considered as a combination of thereservoir and matrix dispersion type systems. In this case, thereservoir of the active substance is formed by first suspending the drugsolids in an aqueous solution of water-soluble polymer and thendispersing the drug suspension in a lipophilic polymer to form amultiplicity of unleachable, microscopic spheres of drug reservoirs.

Any of the herein-described controlled release, extended release, andsustained release compositions can be formulated to release the activeingredient in about 30 minutes to about 1 week, in about 30 minutes toabout 72 hours, in about 30 minutes to 24 hours, in about 30 minutes to12 hours, in about 30 minutes to 6 hours, in about 30 minutes to 4hours, and in about 3 hours to 10 hours. In embodiments, an effectiveconcentration of the active ingredient(s) is sustained in a subject for4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 24 hours, 48hours, 72 hours, or more after administration of the pharmaceuticalcompositions to the subject.

Vaccine or Immunogenic Compositions

The present invention is directed to methods of combination treatment.The combination treatment comprises at least an immunogenic composition,e.g., a neoplasia vaccine or immunogenic composition capable of raisinga specific T-cell response. The neoplasia vaccine or immunogeniccomposition comprises neoantigenic peptides and/or neoantigenicpolypeptides corresponding to tumor specific neoantigens identified bythe methods described herein. The combination treatment also comprisesat least one checkpoint inhibitor. In particular, the present inventionis directed to methods of treating or preventing a neoplasia comprisingthe steps of administering to a subject (a) a neoplasia vaccine orimmunogenic composition, and (b) at least one checkpoint inhibitor.

A suitable neoplasia vaccine or immunogenic composition can preferablycontain a plurality of tumor specific neoantigenic peptides. In anembodiment, the vaccine or immunogenic composition can include between 1and 100 sets of peptides, more preferably between 1 and 50 suchpeptides, even more preferably between 10 and 30 sets peptides, evenmore preferably between 15 and 25 peptides. According to anotherpreferred embodiment, the vaccine or immunogenic composition can includeat least one peptides, more preferably 2, 3, 4, or 5 peptides, Incertain embodiments, the vaccine or immunogenic composition can comprise5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30 different peptides.

The optimum amount of each peptide to be included in the vaccine orimmunogenic composition and the optimum dosing regimen can be determinedby one skilled in the art without undue experimentation. For example,the peptide or its variant may be prepared for intravenous (i.v.)injection, sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c, i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c,i.p. and i.v. For example, doses of between 1 and 500 mg 50 µg and 1.5mg, preferably 10 µg to 500 µg, of peptide or DNA may be given and candepend from the respective peptide or DNA. Doses of this range weresuccessfully used in previous trials (Brunsvig P F, et al., CancerImmunol Immunother. 2006; 55(12): 1553- 1564; M. Staehler, et al., ASCOmeeting 2007; Abstract No 3017). Other methods of administration of thevaccine or immunogenic composition are known to those skilled in theart.

In one embodiment of the present invention the different tumor specificneoantigenic peptides and/or polypeptides arc selected for use in theneoplasia vaccine or immunogenic composition so as to maximize thelikelihood of generating an immune attack against the neoplasia/tumor ofthe patient. Without being bound by theory, it is believed that theinclusion of a diversity of tumor specific neoantigenic peptides cangenerate a broad scale immune attack against a neoplasia/tumor. In oneembodiment, the selected tumor specific neoantigenicpeptides/polypeptides are encoded by missense mutations. In a secondembodiment, the selected tumor specific neoantigenicpeptides/polypeptides are encoded by a combination of missense mutationsand neoORF mutations. In a third embodiment, the selected tumor specificneoantigenic peptides/polypeptides are encoded by neoORF mutations.

In one embodiment in which the selected tumor specific neoantigenicpeptides/polypeptides are encoded by missense mutations, the peptidesand/or polypeptides are chosen based on their capability to associatewith the particular MHC molecules of the patient. Peptides/polypeptidesderived from neoORF mutations can also be selected on the basis of theircapability to associate with the particular MHC molecules of thepatient, but can also be selected even if not predicted to associatewith the particular MHC molecules of the patient.

The vaccine or immunogenic composition is capable of raising a specificcytotoxic T-cells response and/or a specific helper T-cell response.

The vaccine or immunogenic composition can further comprise an adjuvantand/or a carrier. Examples of useful adjuvants and carriers are givenherein herein. The peptides and/or polypeptides in the composition canbe associated with a carrier such as, e.g., a protein or anantigen-presenting cell such as e.g. a dendritic cell (DC) capable ofpresenting the peptide to a T-cell.

Adjuvants are any substance whose admixture into the vaccine orimmunogenic composition increases or otherwise modifies the immuneresponse to the mutant peptide. Carriers are scaffold structures, forexample a polypeptide or a polysaccharide, to which the neoantigenicpeptides, is capable of being associated. Optionally, adjuvants areconjugated covalently or non-covalently to the peptides or polypeptidesof the invention.

The ability of an adjuvant to increase the immune response to an antigenis typically manifested by a significant increase in immune-mediatedreaction, or reduction in disease symptoms. For example, an increase inhumoral immunity is typically manifested by a significant increase inthe titer of antibodies raised to the antigen, and an increase in T-cellactivity is typically manifested in increased cell proliferation, orcellular cytotoxicity, or cytokine secretion. An adjuvant may also alteran immune response, for example, by changing a primarily humoral or Th2response into a primarily cellular, or Th1 response.

Suitable adjuvants include, but are not limited to 1018 ISS, aluminumsalts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF,IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX,Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312,Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174,OM-197-MP-EC, ONTAK, PEPTEL. vector system, PLG microparticles,resiquimod, SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, Aquila’s QS21 stimulon (AquilaBiotech, Worcester, Mass., USA) which is derived from saponin,mycobacterial extracts and synthetic bacterial cell wall mimics, andother proprietary adjuvants such as Ribi’s Detox. Quil or Superfos.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Dupuis M, etal., Cell Immunol. 1998; 186(1): 18-27; Allison A C; Dev Biol Stand.1998; 92:3-11). Also cytokines may be used. Several cytokines have beendirectly linked to influencing dendritic cell migration to lymphoidtissues (e.g., TNF-alpha), accelerating the maturation of dendriticcells into efficient antigen-presenting cells for T-lymphocytes (e.g.,GM-CSF, IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specificallyincorporated herein by reference in its entirety) and acting asimmunoadjuvants (e.g., IL-12) (Gabrilovich D I, et al., J ImmunotherEmphasis Tumor Immunol. 1996 (6):414-418).

Toll like receptors (TLRs) may also be used as adjuvants, and areimportant members of the family of pattern recognition receptors (PRRs)which recognize conserved motifs shared by many micro-organisms, termed“pathogen-associated molecular patterns” (PAMPS). Recognition of these“danger signals” activates multiple elements of the innate and adaptiveimmune system. TLRs are expressed by cells of the innate and adaptiveimmune systems such as dendritic cells (DCs), macrophages, T and Bcells, mast cells, and granulocytes and are localized in differentcellular compartments, such as the plasma membrane, lysosomes,endosomes, and endolysosomes. Different TLRs recognize distinct PAMPS.For example, TLR4 is activated by LPS contained in bacterial cell walls,TLR9 is activated by unmethylated bacterial or viral CpG DNA, and TLR3is activated by double stranded RNA. TLR ligand binding leads to theactivation of one or more intracellular signaling pathways, ultimatelyresulting in the production of many key molecules associated withinflammation and immunity (particularly the transcription factor NF-κBand the Type-1 interferons). TLR mediated DC activation leads toenhanced DC activation, phagocytosis, upregulation of activation andco-stimulation markers such as CD80, CD83, and CD86, expression of CCR7allowing migration of DC to draining lymph nodes and facilitatingantigen presentation to T cells, as well as increased secretion ofcytokines such as type I interferons, IL-12, and IL-6. All of thesedownstream events are critical for the induction of an adaptive immuneresponse.

Among the most promising cancer vaccine or immunogenic compositionadjuvants currently in clinical development are the TLR9 agonist CpG andthe synthetic double-stranded RNA (dsRNA) TLR3 ligand poly-ICLC. Inpreclinical studies poly-ICLC appears to be the most potent TLR adjuvantwhen compared to LPS and CpG due to its induction of pro-inflammatorycytokines and lack of stimulation of IL-10, as well as maintenance ofhigh levels of co-stimulatory molecules in DCs1. Furthermore, poly-ICLCwas recently directly compared to CpG in non-human primates (rhesusmacaques) as adjuvant for a protein vaccine or immunogenic compositionconsisting of human papillomavirus (HPV)16 capsomers (Stahl-Hennig C,Eisenblatter M, Jasny E, et al. Synthetic double-stranded RNAs areadjuvants for the induction of T helper 1 and humoral immune responsesto human papillomavirus in rhesus macaques. PLoS pathogens. April2009;5(4)).

CpG immuno stimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine or immunogenic compositionsetting. Without being bound by theory, CpG oligonucleotides act byactivating the innate (non- adaptive) immune system via Toll-likereceptors (TLR), mainly TLR9. CpG triggered TLR9 activation enhancesantigen-specific humoral and cellular responses to a wide variety ofantigens, including peptide or protein antigens, live or killed viruses,dendritic cell vaccines, autologous cellular vaccines and polysaccharideconjugates in both prophylactic and therapeutic vaccines. Moreimportantly, it enhances dendritic cell maturation and differentiation,resulting in enhanced activation of Thl cells and strong cytotoxic T-lymphocyte (CTL) generation, even in the absence of CD4 T-cell help. TheThl bias induced by TLR9 stimulation is maintained even in the presenceof vaccine adjuvants such as alum or incomplete Freund’s adjuvant (IFA)that normally promote a Th2 bias. CpG oligonucleotides show even greateradjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nano particles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enabled the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Arthur M. Krieg, Nature Reviews, Drug Discovery, 5, Jun.2006,471-484). U.S. Pat. No. 6,406,705 Bl describes the combined use ofCpG oligonucleotides, non-nucleic acid adjuvants and an antigen toinduce an antigen- specific immune response. A commercially availableCpG TLR9 antagonist is dSLIM (double Stem Loop Immunomodulator) byMologen (Berlin, GERMANY), which is a preferred component of thepharmaceutical composition of the present invention. Other TLR bindingmolecules such as RNA binding TLR 7, TLR 8 and/or TLR 9 may also beused.

Other examples of useful adjuvants include, but are not limited to,chemically modified CpGs (e.g. CpR, Idera), Poly(I:C)(e.g. polyi:CI2U),non-CpG bacterial DNA or RNA as well as immunoactive small molecules andantibodies such as cyclophosphamide, sunitinib, bevacizumab, celebrex,NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL-999,CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, andSC58175, which may act therapeutically and/or as an adjuvant. Theamounts and concentrations of adjuvants and additives useful in thecontext of the present invention can readily be determined by theskilled artisan without undue experimentation. Additional adjuvantsinclude colony- stimulating factors, such as Granulocyte MacrophageColony Stimulating Factor (GM-CSF, sargramostim).

Poly-ICLC is a synthetically prepared double-stranded RNA consisting ofpolyI and polyC strands of average length of about 5000 nucleotides,which has been stabilized to thermal denaturation and hydrolysis byserum nucleases by the addition of polylysine andcarboxymethylcellulose. The compound activates TLR3 and the RNAhelicase-domain of MDA5, both members of the PAMP family, leading to DCand natural killer (NK) cell activation and production of a “naturalmix” of type I interferons, cytokines, and chemokines. Furthermore,poly-ICLC exerts a more direct, broad host-targeted anti-infectious andpossibly antitumor effect mediated by the two IFN-inducible nuclearenzyme systems, the 2′5′-OAS and the P1/eIF2a kinase, also known as thePKR (4-6), as well as RIG-I helicase and MDA5.

In rodents and non-human primates, poly-ICLC was shown to enhance T cellresponses to viral antigens, cross-priming, and the induction of tumor-,virus-, and autoantigen-specific CD8+ T-cells. In a recent study innon-human primates, poly-ICLC was found to be essential for thegeneration of antibody responses and T-cell immunity to DC targeted ornon-targeted HIV Gag p24 protein, emphasizing its effectiveness as avaccine adjuvant.

In human subjects, transcriptional analysis of serial whole bloodsamples revealed similar gene expression profiles among the 8 healthyhuman volunteers receiving one single s.c. administration of poly-ICLCand differential expression of up to 212 genes between these 8 subjectsversus 4 subjects receiving placebo. Remarkably, comparison of thepoly-ICLC gene expression data to previous data from volunteersimmunized with the highly effective yellow fever vaccine YF17D showedthat a large number of transcriptional and signal transduction canonicalpathways, including those of the innate immune system, were similarlyupregulated at peak time points.

More recently, an immunologic analysis was reported on patients withovarian, fallopian tube, and primary peritoneal cancer in second orthird complete clinical remission who were treated on a phase 1 study ofsubcutaneous vaccination with synthetic overlapping long peptides (OLP)from the cancer testis antigen NY-ESO-1 alone or with Montanide-1SA-51,or with 1.4 mg poly-ICLC and Montanide. The generation ofNY-ESO-1-specific CD4+ and CD8+ T-cell and antibody responses weremarkedly enhanced with the addition of poly-ICLC and Montanide comparedto OLP alone or OLP and Montanide.

A vaccine or immunogenic composition according to the present inventionmay comprise more than one different adjuvant. Furthermore, theinvention encompasses a therapeutic composition comprising any adjuvantsubstance including any of those herein discussed. It is alsocontemplated that the peptide or polypeptide, and the adjuvant can beadministered separately in any appropriate sequence.

A carrier may be present independently of an adjuvant. The carrier maybe covalently linked to the antigen. A carrier can also be added to theantigen by inserting DNA encoding the carrier in frame with DNA encodingthe antigen. The function of a carrier can for example be to conferstability, to increase the biological activity, or to increase serumhalf-life. Extension of the half-life can help to reduce the number ofapplications and to lower doses, thus are beneficial for therapeutic butalso economic reasons. Furthermore, a carrier may aid presentingpeptides to T-cells. The carrier may be any suitable carrier known tothe person skilled in the art, for example a protein or an antigenpresenting cell. A carrier protein could be but is not limited tokeyhole limpet hemocyanin, serum proteins such as transferrin, bovineserum albumin, human scrum albumin, thyroglobulin or ovalbumin,immunoglobulins, or hormones, such as insulin or palmitic acid. Forimmunization of humans, the carrier may be a physiologically acceptablecarrier acceptable to humans and safe. However, tetanus toxoid and/ordiptheria toxoid are suitable carriers in one embodiment of theinvention. Alternatively, the carrier may be dextrans for examplesepharose.

Cytotoxic T-cells (CTLs) recognize an antigen in the form of a peptidebound to an MHC molecule rather than the intact foreign antigen itself.The MHC molecule itself is located at the cell surface of an antigenpresenting cell. Thus, an activation of CTLs is only possible if atrimeric complex of peptide antigen, MHC molecule, and APC is present.Correspondingly, it may enhance the immune response if not only thepeptide is used for activation of CTLs, but if additionally APCs withthe respective MHC molecule are added. Therefore, in some embodimentsthe vaccine or immunogenic composition according to the presentinvention additionally contains at least one antigen presenting cell.

The antigen-presenting cell (or stimulator cell) typically has an MHCclass I or II molecule on its surface, and in one embodiment issubstantially incapable of itself loading the MHC class I or II moleculewith the selected antigen. As is described in more detail herein, theMHC class I or II molecule may readily be loaded with the selectedantigen in vitro.

CD8+ cell activity may be augmented through the use of CD4+ cells. Theidentification of CD4 T+ cell epitopes for tumor antigens has attractedinterest because many immune based therapies against cancer may be moreeffective if both CD8+ and CD4+ T lymphocytes are used to target apatient’s tumor. CD4+ cells are capable of enhancing CD8 T cellresponses. Many studies in animal models have clearly demonstratedbetter results when both CD4⁺ and CD8⁺ T cells participate in anti-tumorresponses (see e.g., Nishimura et al. (1999) Distinct role ofantigen-specific T helper type 1 (TH1) and Th2 cells in tumoreradication in vivo. J Ex Med 190:617-27). Universal CD4+ T cellepitopes have been identified that are applicable to developingtherapies against different types of cancer (see e.g., Kobayashi et al.(2008) Current Opinion in Immunology 20:221-27). For example, an HLA-DRrestricted helper peptide from tetanus toxoid was used in melanomavaccines to activate CD4+ T cells non-specifically (see e.g., Slingluffet al. (2007) Immunologic and Clinical Outcomes of a Randomized Phase IITrial of Two Multipeptide Vaccines for Melanoma in the Adjuvant Setting,Clinical Cancer Research 13(21):6386-95). It is contemplated within thescope of the invention that such CD4+ cells may be applicable at threelevels that vary in their tumor specificity: 1) a broad level in whichuniversal CD4+ epitopes (e.g., tetanus toxoid) may be used to augmentCD8+ cells; 2) an intermediate level in which native, tumor-associatedCD4+ epitopes may be used to augment CD8+ cells; and 3) a patientspecific level in which neoantigen CD4+ epitopes may be used to augmentCD8+ cells in a patient specific manner.

CD8+ cell immunity may also be generated with neoantigen loadeddendritic cell (DC) vaccine. DCs are potent antigen-presenting cellsthat initiate T cell immunity and can be used as cancer vaccines whenloaded with one or more peptides of interest, for example, by directpeptide injection. For example, patients that were newly diagnosed withmetastatic melanoma were shown to be immunized against 3HLA-A*0201-restricted gp100 melanoma antigen-derived peptides withautologous peptide pulsed CD40L/IFN-g-activated mature DCs via anIL-12p70-producing patient DC vaccine (see e.g., Carreno et al (2013)L-12p70-producing patient DC vaccine elicits Tc1-polarized immunity,Journal of Clinical Investigation, 123(8):3383-94 and Ali et al. (2009)In situ regulation of DC subsets and T cells mediates tumor regressionin mice, Cancer Immunotherapy, 1(8):1-10). It is contemplated within thescope of the invention that neoantigen loaded DCs may be prepared usingthe synthetic TLR 3 agonist Polyinosinic-PolycytidylicAcid-poly-L-lysinc Carboxymethylcellulose (Poly-ICLC) to stimulate theDCs. Poly-ICLC is a potent individual maturation stimulus for human DCsas assessed by an upregulation of CD83 and CD86, induction ofinterleukin-12 (IL-12), tumor necrosis factor (TNF), interferongamma-induced protein 10 (IP-10), interleukin 1 (IL-1), and type Iinterferons (IFN), and minimal interleukin 10 (IL-10) production. DCsmay be differentiated from frozen peripheral blood mononuclear cells(PBMCs) obtained by leukapheresis, while PBMCs may be isolated by Ficollgradient centrifugation and frozen in aliquots.

Illustratively, the following 7 day activation protocol may be used. Day1—PBMCs are thawed and plated onto tissue culture flasks to select formonocytes which adhere to the plastic surface after 1-2 hr incubation at37° C. in the tissue culture incubator. After incubation, thelymphocytes are washed off and the adherent monocytes are cultured for 5days in the presence of interleukin-4 (IL-4) and granulocytemacrophage-colony stimulating factor (GM-CSF) to differentiate toimmature DCs. On Day 6, immature DCs are pulsed with the keyhole limpethemocyanin (KLH) protein which serves as a control for the quality ofthe vaccine and may boost the immunogenicity of the vaccine. The DCs arestimulated to mature, loaded with peptide antigens, and incubatedovernight. On Day 7, the cells are washed, and frozen in 1 ml aliquotscontaining 4-20 x 10(6) cells using a controlled-rate freezer. Lotrelease testing for the batches of DCs may be performed to meet minimumspecifications before the DCs are injected into patients (see e.g.,Sabado et al. (2013) Preparation of tumor antigen-loaded maturedendritic cells for immunotherapy, J. Vis Exp. Aug 1;(78). doi:10.3791/50085).

A DC vaccine may be incorporated into a scaffold system to facilitatedelivery to a patient. Therapeutic treatment of a patients neoplasiawith a DC vaccine may utilize a biomaterial system that releases factorsthat recruit host dendritic cells into the device, differentiates theresident, immature DCs by locally presenting adjuvants (e.g., dangersignals) while releasing antigen, and promotes the release of activated,antigen loaded DCs to the lymph nodes (or desired site of action) wherethe DCs may interact with T cells to generate a potent cytotoxic Tlymphocyte response to the cancer neoantigens. Implantable biomaterialsmay be used to generate a potent cytotoxic T lymphocyte response againsta neoplasia in a patient specific manner. The biomaterial-residentdendritic cells may then be activated by exposing them to danger signalsmimicking infection, in concert with release of antigen from thebiomaterial. The activated dendritic cells then migrate from thebiomaterials to lymph nodes to induce a cytotoxic T effector response.This approach has previously been demonstrated to lead to regression ofestablished melanoma in preclinical studies using a lysate prepared fromtumor biopsies (see e.g., Ali et al. (2209) In situ regulation of DCsubsets and T cells mediates tumor regression in mice, CancerImmunotherapy 1(8):1-10; Ali et al. (2009) Infection-mimicking materialsto program dendritic cells in situ. Nat Mater 8:151-8), and such avaccine is currently being tested in a Phase I clinical trial recentlyinitiated at the Dana-Farber Cancer Institute. This approach has alsobeen shown to lead to regression of glioblastoma, as well as theinduction of a potent memory response to prevent relapse, using the C6rat glioma model.24 in the current proposal. The ability of such animplantable, biomatrix vaccine delivery scaffold to amplify and sustaintumor specific dendritic cell activation may lead to more robustanti-tumor immunosensitization than can be achieved by traditionalsubcutaneous or intra-nodal vaccine administrations.

Preferably, the antigen presenting cells are dendritic cells. Suitably,the dendritic cells are autologous dendritic cells that are pulsed withthe neoantigenic peptide. The peptide may be any suitable peptide thatgives rise to an appropriate T-cell response. T-cell therapy usingautologous dendritic cells pulsed with peptides from a tumor associatedantigen is disclosed in Murphy et al. (1996) The Prostate 29, 371-380and Tjua et al. (1997) The Prostate 32, 272-278.

Thus, in one embodiment of the present invention the vaccine orimmunogenic composition containing at least one antigen presenting cellis pulsed or loaded with one or more peptides of the present invention.Alternatively, peripheral blood mononuclear cells (PBMCs) isolated froma patient may be loaded with peptides ex vivo and injected back into thepatient. As an alternative the antigen presenting cell comprises anexpression construct encoding a peptide of the present invention. Thepolynucleotide may be any suitable polynucleotide and it is preferredthat it is capable of transducing the dendritic cell, thus resulting inthe presentation of a peptide and induction of immunity.

The inventive pharmaceutical composition may be compiled so that theselection, number and/or amount of peptides present in the compositionis/are tissue, cancer, and/or patient-specific. For instance, the exactselection of peptides can be guided by expression patterns of the parentproteins in a given tissue to avoid side effects. The selection may bedependent on the specific type of cancer, the status of the disease,earlier treatment regimens, the immune status of the patient, and, ofcourse, the HLA-haplotypc of the patient. Furthermore, the vaccine orimmunogenic composition according to the invention can containindividualized components, according to personal needs of the particularpatient. Examples include varying the amounts of peptides according tothe expression of the related neoantigen in the particular patient,unwanted side-effects due to personal allergies or other treatments, andadjustments for secondary treatments following a first round or schemeof treatment.

Pharmaceutical compositions comprising the peptide of the invention maybe administered to an individual already suffering from cancer. Intherapeutic applications, compositions are administered to a patient inan amount sufficient to elicit an effective CTL response to the tumorantigen and to cure or at least partially arrest symptoms and/orcomplications. An amount adequate to accomplish this is defined as“therapeutically effective dose.” Amounts effective for this use candepend on, e.g., the peptide composition, the manner of administration,the stage and severity of the disease being treated, the weight andgeneral state of health of the patient, and the judgment of theprescribing physician, but generally range for the initial immunization(that is for therapeutic or prophylactic administration) from about 1.0µg to about 50,000 µg of peptide for a 70 kg patient, followed byboosting dosages or from about 1.0 µg to about 10,000 µg of peptidepursuant to a boosting regimen over weeks to months depending upon thepatient’s response and condition and possibly by measuring specific CTLactivity in the patient’s blood. It should be kept in mind that thepeptide and compositions of the present invention may generally beemployed in serious disease states, that is, life-threatening orpotentially life threatening situations, especially when the cancer hasmetastasized. For therapeutic use, administration should begin as soonas possible after the detection or surgical removal of tumors. This isfollowed by boosting doses until at least symptoms are substantiallyabated and for a period thereafter.

The pharmaceutical compositions (e.g., vaccine compositions) fortherapeutic treatment are intended for parenteral, topical, nasal, oralor local administration. Preferably, the pharmaceutical compositions areadministered parenterally, e.g., intravenously, subcutaneously,intradermally, or intramuscularly. The compositions may be administeredat the site of surgical excision to induce a local immune response tothe tumor. The invention provides compositions for parenteraladministration which comprise a solution of the peptides and vaccine orimmunogenic compositions are dissolved or suspended in an acceptablecarrier, preferably an aqueous carrier. A variety of aqueous carriersmay be used, e.g., water, buffered water, 0.9% saline, 0.3% glycine,hyaluronic acid and the like. These compositions may be sterilized byconventional, well known sterilization techniques, or may be sterilefiltered. The resulting aqueous solutions may be packaged for use as is,or lyophilized, the lyophilized preparation being combined with asterile solution prior to administration. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, etc.

A liposome suspension containing a peptide may be administeredintravenously, locally, topically, etc. in a dose which varies accordingto, inter alia, the manner of administration, the peptide beingdelivered, and the stage of the disease being treated. For targeting tothe immune cells, a ligand, such as, e.g., antibodies or fragmentsthereof specific for cell surface determinants of the desired immunesystem cells, can be incorporated into the liposome.

For solid compositions, conventional or nanoparticle nontoxic solidcarriers may be used which include, for example, pharmaceutical gradesof mannitol, lactose, starch, magnesium stearate, sodium saccharin,talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.For oral administration, a pharmaceutically acceptable nontoxiccomposition is formed by incorporating any of the normally employedexcipients, such as those carriers previously listed, and generally10-95% of active ingredient, that is, one or more peptides of theinvention, and more preferably at a concentration of 25%-75%.

For aerosol administration, the immunogenic peptides are preferablysupplied in finely divided form along with a surfactant and propellant.Typical percentages of peptides are 0.01%-20% by weight, preferably1%-10%. The surfactant can, of course, be nontoxic, and preferablysoluble in the propellant. Representative of such agents are the estersor partial esters of fatty acids containing from 6 to 22 carbon atoms,such as caproic, octanoic, lauric, palmitic, stearic, linoleic,linolenic, olesteric and oleic acids with an aliphatic polyhydricalcohol or its cyclic anhydride. Mixed esters, such as mixed or naturalglycerides may be employed. The surfactant may constitute 0.1%-20% byweight of the composition, preferably 0.25-5%. The balance of thecomposition is ordinarily propellant. A carrier can also be included asdesired, as with, e.g., lecithin for intranasal delivery.

The peptides and polypeptides of the invention can be readilysynthesized chemically utilizing reagents that are free of contaminatingbacterial or animal substances (Merrifield RB: Solid phase peptidesynthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc.85:2149-54, 1963).

The peptides and polypeptides of the invention can also be expressed bya vector, e.g., a nucleic acid molecule as herein-discussed, e.g., RNAor a DNA plasmid, a viral vector such as a poxvirus, e.g., orthopoxvirus, avipox virus, or adenovirus, AAV or lentivirus. This approachinvolves the use of a vector to express nucleotide sequences that encodethe peptide of the invention. Upon introduction into an acutely orchronically infected host or into a noninfected host, the vectorexpresses the immunogenic peptide, and thereby elicits a host CTLresponse.

For therapeutic or immunization purposes, nucleic acids encoding thepeptide of the invention and optionally one or more of the peptidesdescribed herein can also be administered to the patient. A number ofmethods are conveniently used to deliver the nucleic acids to thepatient. For instance, the nucleic acid can be delivered directly, as“naked DNA”. This approach is described, for instance, in Wolff et al.,Science 247: 1465-1468 (1990) as well as U.S. Pat. Nos. 5,580,859 and5,589,466. The nucleic acids can also be administered using ballisticdelivery as described, for instance, in U.S. Patent No. 5,204,253.Particles comprised solely of DNA can be administered. Alternatively,DNA can be adhered to particles, such as gold particles. Generally, aplasmid for a vaccine or immunological composition can comprise DNAencoding an antigen (e.g., one or more neoantigens) operatively linkedto regulatory sequences which control expression or expression andsecretion of the antigen from a host cell, e.g., a mammalian cell; forinstance, from upstream to downstream, DNA for a promoter, such as amammalian virus promoter (e.g., a CMV promoter such as an hCMV or mCMVpromoter, e.g., an early-intermediate promoter, or an SV40 promoter--seedocuments cited or incorporated herein for useful promoters), DNA for aeukaryotic leader peptide for secretion (e.g., tissue plasminogenactivator), DNA for the neoantigen(s), and DNA encoding a terminator(e.g., the 3′ UTR transcriptional terminator from the gene encodingBovine Growth Hormone or bGH polyA). A composition can contain more thanone plasmid or vector, whereby each vector contains and expresses adifferent neoantigen. Mention is also made of Wasmoen U.S. Pat. No.5,849,303, and Dale U.S. Pat. No. 5,811,104, whose text may be useful.DNA or DNA plasmid formulations can be formulated with or insidecationic lipids; and, as to cationic lipids, as well as adjuvants,mention is also made of Loosmore U.S. Pat. Application 2003/0104008.Also, teachings in Audonnet U.S. Pat. Nos. 6,228,846 and 6,159,477 maybe relied upon for DNA plasmid teachings that can be employed inconstructing and using DNA plasmids that contain and express in vivo.

The nucleic acids can also be delivered complexed to cationic compounds,such as cationic lipids. Lipid-mediated gene delivery methods aredescribed, for instance, in WO1996/18372; WO 1993/24640; Mannino &Gould-Fogerite , BioTechniques 6(7): 682-691 (1988); U.S. Pat. No.5,279,833; WO 1991/06309; and Feigner et al., Proc. Natl. Acad. Sci. USA84: 7413-7414 (1987).

RNA encoding the peptide of interest (e.g., mRNA) can also be used fordelivery (see, e.g., Kiken et al, 2011; Su et al , 2011; see also US8278036; Halabi et al. J Clin Oncol (2003) 21:1232-1237; Petsch et al,Nature Biotechnology 2012 Dec 7;30(12):1210-6).

Information concerning poxviruses that may be used in the practice ofthe invention, such as Chordopoxvirinae subfamily poxviruses (poxvirusesof vertebrates), for instance, orthopoxviruses and avipoxviruses, e.g.,vaccinia virus (e.g., Wyeth Strain, WR Strain (e.g., ATCC® VR-1354),Copenhagen Strain, NYVAC, NYVAC.1, NYVAC.2, MVA, MVA-BN), canarypoxvirus (e.g., Wheatley C93 Strain, ALVAC), fowlpox virus (e.g., FP9Strain, Webster Strain, TROVAC), dovepox, pigeonpox, quailpox, andraccoon pox, inter alia, synthetic or non-naturally occurringrecombinants thereof, uses thereof, and methods for making and usingsuch recombinants may be found in scientific and patent literature, suchas:

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each of which is incorporated herein by reference.

As to adenovirus vectors useful in the practice of the invention,mention is made of U.S. Pat. No. 6,955,808. The adenovirus vector usedcan be selected from the group consisting of the Ad5, Ad35, Ad11, C6,and C7 vectors. The sequence of the Adenovirus 5 (“Ad5”) genome has beenpublished. (Chroboczek, J., Bieber, F., and Jacrot, B. (1992) TheSequence of the Genome of Adenovirus Type 5 and Its Comparison with theGenome of Adenovirus Type 2, Virology 186, 280-285; the contents ifwhich is hereby incorporated by reference). Ad35 vectors are describedin U.S. Pat. Nos. 6,974,695, 6,913,922, and 6,869,794. Ad11 vectors aredescribed in U.S. Pat. No. 6,913,922. C6 adenovirus vectors aredescribed in U.S. Pat. Nos. 6,780,407; 6,537,594; 6,309,647; 6,265,189;6,156,567; 6,090,393; 5,942,235 and 5,833,975. C7 vectors are describedin U.S. Pat. No. 6,277,558. Adenovirus vectors that arc E1-defective ordeleted, E3-defective or deleted, and/or F4-defective or deleted mayalso be used. Certain adenoviruses having mutations in the E1 regionhave improved safety margin because E1-defective adenovirus mutants arereplication-defective in non-permissive cells, or, at the very least,are highly attenuated. Adenoviruses having mutations in the E3 regionmay have enhanced the immunogenicity by disrupting the mechanism wherebyadenovirus down-regulates MHC class I molecules. Adenoviruses having E4mutations may have reduced immunogenicity of the adenovirus vectorbecause of suppression of late gene expression. Such vectors may beparticularly useful when repeated re-vaccination utilizing the samevector is desired. Adenovirus vectors that are deleted or mutated in E1,E3, E4, E1 and E3, and E1 and E4 can be used in accordance with thepresent invention. Furthermore, “gutless” adenovirus vectors, in whichall viral genes are deleted, can also be used in accordance with thepresent invention. Such vectors require a helper virus for theirreplication and require a special human 293 cell line expressing bothE1a and Cre, a condition that does not exist in natural environment.Such “gutless” vectors are non-immunogenic and thus the vectors may beinoculated multiple times for re-vaccination. The “gutless” adenovirusvectors can be used for insertion of heterologous inserts/genes such asthe transgenes of the present invention, and can even be used forco-delivery of a large number of heterologous inserts/genes.

As to lentivirus vector systems useful in the practice of the invention,mention is made of U.S. Pats. Nos. 6428953, 6165782, 6013516, 5994136,6312682, and 7,198,784, and documents cited therein.

With regard to AAV vectors useful in the practice of the invention,mention is made of U.S. Pat. Nos. 5658785, 7115391, 7172893, 6953690,6936466, 6924128, 6893865, 6793926, 6537540, 6475769 and 6258595, anddocuments cited therein.

Another vector is BCG (Bacille Calmette Guerin). BCG vectors aredescribed in Stover et al. (Nature 351:456-460 (1991)). A wide varietyof other vectors useful for therapeutic administration or immunizationof the peptides of the invention, e.g., Salmonella typhi vectors and thelike, is apparent to those skilled in the art from the descriptionherein.

Vectors can be administered so as to have in vivo expression andresponse akin to doses and/or responses elicited by antigenadministration

A preferred means of administering nucleic acids encoding the peptide ofthe invention uses minigene constructs encoding multiple epitopes. Tocreate a DNA sequence encoding the selected CTL epitopes (minigene) forexpression in human cells, the amino acid sequences of the epitopes arereverse translated. A human codon usage table is used to guide the codonchoice for each amino acid. These epitope-encoding DNA sequences aredirectly adjoined, creating a continuous polypeptide sequence. Tooptimize expression and/or immunogenicity, additional elements can beincorporated into the minigene design. Examples of amino acid sequencethat could be reverse translated and included in the minigene sequenceinclude: helper T lymphocyte, epitopes, a leader (signal) sequence, andan endoplasmic reticulum retention signal. In addition, MHC presentationof CTL epitopes may be improved by including synthetic (e.g.poly-alanine) or naturally- occurring flanking sequences adjacent to theCTL epitopes.

The minigene sequence is converted to DNA by assembling oligonucleotidesthat encode the plus and minus strands of the minigene. Overlappingoligonucleotides (30-100 bases long) are synthesized, phosphorylated,purified and annealed under appropriate conditions using well knowntechniques. The ends of the oligonucleotides are joined using T4 DNAligase. This synthetic minigene, encoding the CTL epitope polypeptide,can then cloned into a desired expression vector.

Standard regulatory sequences well known to those of skill in the artare included in the vector to ensure expression in the target cells.Several vector elements are required: a promoter with a down-streamcloning site for minigene insertion; a polyadenylation signal forefficient transcription termination; an E. coli origin of replication;and an E. coli selectable marker (e.g. ampicillin or kanamycinresistance). Numerous promoters can be used for this purpose, e.g., thehuman cytomegalovirus (hCMV) promoter. See, U.S. Pat. Nos. 5,580,859 and5,589,466 for other suitable promoter sequences.

Additional vector modifications may be desired to optimize minigeneexpression and immunogenicity. In some cases, introns are required forefficient gene expression, and one or more synthetic ornaturally-occurring introns could be incorporated into the transcribedregion of the minigene. The inclusion of mRNA stabilization sequencescan also be considered for increasing minigene expression. It hasrecently been proposed that immuno stimulatory sequences (ISSs or CpGs)play a role in the immunogenicity of DNA’ vaccines. These sequencescould be included in the vector, outside the minigene coding sequence,if found to enhance immunogenicity.

In some embodiments, a bicistronic expression vector, to allowproduction of the minigene-encoded epitopes and a second proteinincluded to enhance or decrease immunogenicity can be used. Examples ofproteins or polypeptides that could beneficially enhance the immuneresponse if co-expressed include cytokines (e.g., IL2, IL12, GM-CSF),cytokine-inducing molecules (e.g. LeIF) or costimulatory molecules.Helper (HTL) epitopes could be joined to intracellular targeting signalsand expressed separately from the CTL epitopes. This would allowdirection of the EiTL epitopes to a cell compartment different than theCTL epitopes. If required, this could facilitate more efficient entry ofHTL epitopes into the MHC class II pathway, thereby improving CTLinduction. In contrast to CTL induction, specifically decreasing theimmune response by co-expression of immunosuppressive molecules (e.g.TGF-β) may be beneficial in certain diseases.

Once an expression vector is selected, the minigene is cloned into thepolylinker region downstream of the promoter. This plasmid istransformed into an appropriate E. coli strain, and DNA is preparedusing standard techniques. The orientation and DNA sequence of theminigene, as well as all other elements included in the vector, areconfirmed using restriction mapping and DNA sequence analysis. Bacterialcells harboring the correct plasmid can be stored as a master cell bankand a working cell bank.

Purified plasmid DNA can be prepared for injection using a variety offormulations. The simplest of these is reconstitution of lyophilized DNAin sterile phosphate-buffer saline (PBS). A variety of methods have beendescribed, and new techniques may become available. As noted herein,nucleic acids are conveniently formulated with cationic lipids. Inaddition, glycolipids, fusogenic liposomes, peptides and compoundsreferred to collectively as protective, interactive, non-condensing(PINC) could also be complexed to purified plasmid DNA to influencevariables such as stability, intramuscular dispersion, or trafficking tospecific organs or cell types.

Target cell sensitization can be used as a functional assay forexpression and MHC class I presentation of minigene-encoded CTLepitopes. The plasmid DNA is introduced into a mammalian cell line thatis suitable as a target for standard CTL. chromium release assays. Thetransfection method used is dependent on the final formulation.Electroporation can be used for “naked” DNA, whereas cationic lipidsallow direct in vitro transfection. A plasmid expressing greenfluorescent protein (GFP) can be co-transfcctcd to allow enrichment oftransfected cells using fluorescence activated cell sorting (FACS).These cells are then chromium-51 labeled and used as target cells forepitope- specific CTL lines. Cytolysis, detected by 51 Cr release,indicates production of MHC presentation of mini gene-encoded CTLepitopes.

In vivo immunogenicity is a second approach for functional testing ofminigene DNA formulations. Transgenic mice expressing appropriate humanMHC molecules are immunized with the DNA product. The dose and route ofadministration are formulation dependent (e.g. IM for DNA in PBS, IP forlipid-complexed DNA). Twenty-one days after immunization, splenocytesare harvested and restimulated for 1 week in the presence of peptidesencoding each epitope being tested. These effector cells (CTLs) areassayed for cytolysis of peptide-loaded, chromium-51 labeled targetcells using standard techniques. Lysis of target cells sensitized by MHCloading of peptides corresponding to minigene-encoded epitopesdemonstrates DNA vaccine function for in vivo induction of CTLs.

Peptides may be used to elicit CTL ex vivo, as well. The resulting CTL,can be used to treat chronic tumors in patients in need thereof that donot respond to other conventional forms of therapy, or does not respondto a peptide vaccine approach of therapy. Ex vivo CTL responses to aparticular tumor antigen arc induced by incubating in tissue culture thepatient’s CTL precursor cells (CTLp) together with a source ofantigen-presenting cells (APC) and the appropriate peptide. After anappropriate incubation time (typically 1-4 weeks), in which the CTLp areactivated and mature and expand into effector CTL, the cells are infusedback into the patient, where they destroy their specific target cell(i.e., a tumor cell). In order to optimize the in vitro conditions forthe generation of specific cytotoxic T cells, the culture of stimulatorcells are maintained in an appropriate serum-free medium.

Prior to incubation of the stimulator cells with the cells to beactivated, e.g., precursor CD8+ cells, an amount of antigenic peptide isadded to the stimulator cell culture, of sufficient quantity to becomeloaded onto the human Class I molecules to be expressed on the surfaceof the stimulator cells. In the present invention, a sufficient amountof peptide is an amount that allows about 200, and preferably 200 ormore, human Class I MHC molecules loaded with peptide to be expressed onthe surface of each stimulator cell. Preferably, the stimulator cellsare incubated with >2 µg/ml peptide. For example, the stimulator cellsare incubates with > 3, 4, 5, 10, 15, or more µg/ml peptide.

Resting or precursor CD8+ cells are then incubated in culture with theappropriate stimulator cells for a time period sufficient to activatethe CD8+ cells. Preferably, the CD8+ cells are activated in an antigen-specific manner. The ratio of resting or precursor CD8+. (effector)cells to stimulator cells may vary from individual to individual and mayfurther depend upon variables such as the amenability of an individual’slymphocytes to culturing conditions and the nature and severity of thedisease condition or other condition for which the within-describedtreatment modality is used. Preferably, however, the lymphocyte:stimulator cell ratio is in the range of about 30: 1 to 300: 1. Theeffector/stimutator culture may be maintained for as long a time as isnecessary to stimulate a therapeutically useable or effective number ofCD8+ cells.

The induction of CTL in vitro requires the specific recognition ofpeptides that are bound to allele specific MHC class I molecules on APC.The number of specific MHC/peptide complexes per APC is crucial for thestimulation of CTL, particularly in primary immune responses. Whilesmall amounts of peptide/MHC complexes per cell are sufficient to rendera cell susceptible to lysis by CTL, or to stimulate a secondary CTLresponse, the successful activation of a CTL precursor (pCTL) duringprimary response requires a significantly higher number of MHC/peptidecomplexes. Peptide loading of empty major histocompatability complexmolecules on cells allows the induction of primary cytotoxic ‘1’lymphocyte responses.

Since mutant cell lines do not exist for every human MHC allele, it isadvantageous to use a technique to remove endogenous MHC- associatedpeptides from the surface of APC, followed by loading the resultingempty MHC molecules with the immunogenic peptides of interest. The useof non-transformed (non-tumorigenic), noninfected cells, and preferably,autologous cells of patients as APC is desirable for the design of CTLinduction protocols directed towards development of ex vivo CTLtherapies. This application discloses methods for stripping theendogenous MHC-associated peptides from the surface of APC followed bythe loading of desired peptides.

A stable MHC: class I molecule is a trimeric complex formed of thefollowing elements: 1) a peptide usually of 8 - 10 residues, 2) atransmembrane heavy polymorphic protein chain which bears thepeptide-binding site in its a1 and a2 domains, and 3) a non-covalentlyassociated non-polymorphic light chain, p2microglobuiin. Removing thebound peptides and/or dissociating the p2microglobulin from the complexrenders the MHC class I molecules nonfunctional and unstable, resultingin rapid degradation. All MHC class I molecules isolated from PBMCs haveendogenous peptides bound to them. Therefore, the first step is toremove all endogenous peptides bound to MHC class 1 molecules on the APCwithout causing their degradation before exogenous peptides can be addedto them.

Two possible ways to free up MHC class I molecules of bound peptidesinclude lowering the culture temperature from 37° C. to 26° C. overnightto destablize p2microglobulin and stripping the endogenous peptides fromthe cell using a mild acid treatment. The methods release previouslybound peptides into the extracellular environment allowing new exogenouspeptides to bind to the empty class I molecules. The cold-temperatureincubation method enables exogenous peptides to bind efficiently to theMHC complex, but requires an overnight incubation at 26° C. which mayslow the cell’s metabolic rate. It is also likely that cells notactively synthesizing MHC molecules (e.g., resting PBMC) would notproduce high amounts of empty surface MHC molecules by the coldtemperature procedure.

Harsh acid stripping involves extraction of the peptides withtrifluoroacetic acid, pH 2, or acid denaturation of the immunoaffinitypurified class I-peptide complexes. These methods are not feasible forCTL induction, since it is important to remove the endogenous peptideswhile preserving APC viability and an optimal metabolic state which iscritical for antigen presentation. Mild acid solutions of pH 3 such asglycine or citrate -phosphate buffers have been used to identifyendogenous peptides and to identify tumor associated T cell epitopes.The treatment is especially effective, in that only the MHC class Imolecules are destabilized (and associated peptides released), whileother surface antigens remain intact, including MHC class II molecules.Most importantly, treatment of cells with the mild acid solutions do notaffect the cell’s viability or metabolic state. The mild acid treatmentis rapid since the stripping of the endogenous peptides occurs in twominutes at 4° C. and the APC is ready to perform its function after theappropriate peptides are loaded. The technique is utilized herein tomake peptide-specific APCs for the generation of primary antigen-specific CTL. The resulting APC are efficient in inducing peptide-specific CD8+ CTL.

Activated CD8+ cells may be effectively separated from the stimulatorcells using one of a variety of known methods. For example, monoclonalantibodies specific for the stimulator cells, for the peptides loadedonto the stimulator cells, or for the CD8+ cells (or a segment thereof)may be utilized to bind their appropriate complementary ligand.Antibody-tagged molecules may then be extracted from thestimulator-effector cell admixture via appropriate means, e.g., viawell-known immunoprecipitation or immunoassay methods.

Effective, cytotoxic amounts of the activated CD8+ cells can varybetween in vitro and in vivo uses, as well as with the amount and typeof cells that are the ultimate target of these killer cells. The amountcan also vary depending on the condition of the patient and should bedetermined via consideration of all appropriate factors by thepractitioner. Preferably, however, about 1 × 10⁶ to about 1 × 10¹², morepreferably about 1 × 10⁸ to about 1 × 10¹¹, and even more preferably,about 1 × 10⁹ to about 1 × 10¹⁰ activated CD8+ cells are utilized foradult humans, compared to about 5 × 10⁶ - 5 × 10⁷ cells used in mice.

Preferably, as discussed herein, the activated CD8+ cells are harvestedfrom the cell culture prior to administration of the CD8+ cells to theindividual being treated. It is important to note, however, that unlikeother present and proposed treatment modalities, the present method usesa cell culture system that is not tumorigenic. Therefore, if completeseparation of stimulator cells and activated CD8+ cells are notachieved, there is no inherent danger known to be associated with theadministration of a small number of stimulator cells, whereasadministration of mammalian tumor-promoting cells may be extremelyhazardous.

Methods of re-introducing cellular components arc known in the art andinclude procedures such as those exemplified in U.S. Pat. No. 4,844,893to Honsik, et al. and U.S. Pat. No. 4,690,915 to Rosenberg. For example,administration of activated CD8+ cells via intravenous infusion isappropriate.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are well within the purview of the skilled artisan.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook,1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culturc”(Freshney, 1987); “Methods in Enzymology” “Handbook of ExperimentalImmunology” (Wei, 1996); “Gene Transfer Vectors for Mammalian Cells”(Miller and Calos, 1987); “Current Protocols in Molecular Biology”(Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994);“Current Protocols in Immunology” (Coligan, 1991). These techniques areapplicable to the production of the polynucleotides and polypeptides ofthe invention, and, as such, may be considered in making and practicingthe invention. Particularly useful techniques for particular embodimentsarc discussed in the sections that follow.

Therapeutic Methods

The present invention provides methods of inducing a neoplasia/tumorspecific immune response in a subject, vaccinating against aneoplasia/tumor, treating and or alleviating a symptom of cancer in asubject by administering the subject a neoplasia vaccine or aneoantigenic peptide or composition of the invention and at least onecheckpoint inhibitor.

In particular, the present invention is directed to methods of treatingor preventing a neoplasia comprising the steps of administering to asubject (a) a neoplasia vaccine or immunogenic composition, and (b) atleast one checkpoint inhibitor.

According to the invention, the herein-described neoplasia vaccine orimmunogenic composition may be used for a patient that has beendiagnosed as having cancer, or at risk of developing cancer.

The described combination of the invention is administered in an amountsufficient to induce a CTL response.

Additional Therapies

The tumor specific neoantigen peptides and pharmaceutical compositionsdescribed herein can also be administered in further combination withanother agent, for example a therapeutic agent. In certain embodiments,the additional agents can be, but arc not limited to, chemotherapeuticagents, anti-angiogenesis agents and agents that reduceimmune-suppression.

The neoplasia vaccine or immunogenic composition and one or morecheckpoint inhibitors can be administered before, during, or afteradministration of the additional agent. In embodiments, the neoplasiavaccine or immunogenic composition and/or one or more checkpointinhibitors are administered before the first administration of theadditional agent. In other embodiments, the neoplasia vaccine orimmunogenic composition and/or one or more checkpoint inhibitors areadministered after the first administration of the additionaltherapeutic agent (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14days or more). In embodiments, the neoplasia vaccine or immunogeniccomposition and one or more checkpoint inhibitors are administeredsimultaneously with the first administration of the additionaltherapeutic agent.

The therapeutic agent is for example, a chemotherapeutic orbiotherapeutic agent, radiation, or immunotherapy. Any suitabletherapeutic treatment for a particular cancer may be administered.Examples of chemotherapeutic and biotherapeutic agents include, but arenot limited to, an angiogenesis inhibitor, such ashydroxy angiostatinK1-3, DL-α-Difluoromethyl-ornithinc, cndostatin, fumagillin, genistein,minocycline, staurosporine, and thalidomide; a DNAintercaltor-cross-linker, such as Bleomycin, Carboplatin, Carmustine,Chlorambucil, Cyclophosphamide, cis-Diammineplatinum(II) dichloride(Cisplatin), Melphalan, Mitoxantrone, and Oxaliplatin; a DNA synthesisinhibitor, such as (±)-Amethupterin (Methotrexate),3-Amino-1,2,4-benzotriazine 1,4-dioxide, Aminopterin, Cytosineβ-D-arabinofuranoside, 5-Fluoro-5′-deoxyuridine, 5-Fluorouracil,Ganciclovir, Hydroxyurea, and Mitomycin C; a DNA-RNA transcriptionregulator, such as Actinomycin D, Daunorubicin, Doxorubicin,Homoharringtonine, and Idarubicin; an enzyme inhibitor, such asS(+)-Camptothecin, Curcumin, (-)-Deguelin, 5,6-Dichlorobenzimidazole1-β-D-ribofuranoside, Etoposide. Formestane, Fostriecin, Hispidin,2-Imino-1-imidar.oli-dineacetic acid (Cyclocreatine), Mevinolin.Trichostatin A, Tyrphostin AG 34, and Tyrphostin AG 879; a generegulator, such as 5-Aza-2′-deoxycytidine, 5-Azacytidine,Cholecalcifcrol (Vitamin D3), 4-Hydroxytamoxifen, Melatonin,Mifcpristonc, Raloxifene, all trans-Retinal (Vitamin A aldehyde).Retinoic acid all trans (Vitamin A acid), 9-cis-Retinoic Acid,13-cis-Retinoic acid, Retinol (Vitamin A), Tamoxifen, and Troglitazone;a microtubule inhibitor, such as Colchicine, docetaxel, Dolastatin 15,Nocodazole, Paclitaxel, Podophyllotoxin, Rhizoxin, Vinblastine,Vincristine, Vindesine, and Vinorclbine (Navelbinc); and an unclassifiedtherapeutic agent, such as 17-(Allylamino)-17-dernethoxygeldananiycin,4-Arnino-1,8-naphthalimide, Apigenin, Brefeldin A, Cimetidine,Dichloromethylene-diphosphonic acid, Leuprolide (Leuprorelin),Luteinizing Hormone-Releasing Hormone, Pifithrin-a, Rapamycin, Sexhormone-binding globulin, Thapsigargin, and Urinary trypsin inhibitorfragment (Bikunin). The therapeutic agent may be altretamine,amifostine, asparaginase, capecitabine, cladribine, cisapride,cytarabine, dacarbazine (DTIC), dactinomycin, dronabinol, epoetin alpha,filgrastim, fludarabine, gemcitabine, granisetron, ifosfamide,irinotecan, lansoprazole, levamisolc, leucovorin, megestrol, mesna,metoclopramide, mitotane, omeprazole, ondansetron, pilocarpine,prochloroperazine, or topotecan hydrochloride. The therapeutic agent maybe a monoclonal antibody such as rituximab (Rituxan®), alemtuzumab(Campath®), Bevacizumab (Avastin®), Cetuximab (Erbitux®), panitumumab(Vectibix®), and trastuzumab (Herceptin®), Vemurafenib (Zelboraf®)imatinib mesylate (Gleevec®), erlotinib (Tarceva′®), gefitinib(Iressa®), Vismodegib (Erivedge™), 90Y-ibritumomab tiuxetan,131I-tositumomab, ado-trastuzumab emtansine, lapatinib (Tykerb®),pertuzumab (Perjeta™), ado-trastuzumab emtansine (Kadeyla™), regorafenib(Stivarga®), sunitinib (Sutent®), Denosumab (Xgeva®), sorafenib(Nexavar®), pazopanib (Votrient®), axitinib (Inlyta®), dasatinib(Sprycel®), nilotinib (Tasigna®), bosutinib (Bosulif®), ofatumumab(Arzerra®), obinutuzumab (Gazyva™), ibrutinib (Imbruvica™), idelalisib(Zydeligt®), crizotinib (Xalkori®, erlotinib (Tarceva®), atatinibdimaleate (Gilotrif®), ceritinib (LDK378/Zykadia), Tositumomab and131I-tusiturnumab (Bexxar®), ibritumomab tiuxetan (Zevalin®),brentuximab vedotin (Adcetris®), bortezomib (Velcade®), siltuximab(Sylvant™), trametinib (Mekinist®), dabrafenib (Tafinlar®),pembrolizumab (Keytruda®), carfilzomib (Kyprolis®), Ramucirumab(Cyramza™), Cabozantinib (Cometriq™), vandetanib (Caprelsa®),Optionally, the therapeutic agent is a neoantigen. The therapeutic agentmay be a cytokine such as interferons (INFs), interleukins (ILs), orhematopoietic growth factors. The therapeutic agent may be INF-α, IL-2,Aldesleukin, IL-2, Erythropoietin, Granulocyte-macrophagecolony-stimulating factor (GM-CSF) or granulocyte colony-stimulatingfactor. The therapeutic agent may be a targeted therapy such astoremifene (Fareston®), fulvestrant (Faslodex®), anastrozole(Arimidex®), exemestane (Aromasin®), letrozole (Femara®),ziv-aflibcrcept (Zaltrap®), Alitretinoin (Panretin®), temsirolimus(Torisel®), Tretinoin (Vesanoid®), denileukin diftitox (Ontak®),vorinostat (Zolinza®), romidepsin (Istodax®), bexarotene (Targretin®),pralatrexate (Folotyn®), lenaliomide (Revlimid®), belinostat(Beleodaq™), lenaliomide (Revlimid®), pomalidomide (Pomalyst®),Cabazitaxel (Jevtana®), enzalutamide (Xtandi®), abiraterone acetate(Zytiga®), radium 223 chloride (Xofigo®), or everolimus (Afinitor®).Aditionally, the therapeutic agent may be an epigenetic targeted drugsuch as HDAC inhibitors, kinase inhibitors, DNA methyltransferaseinhibitors, histone demethylase inhibitors, or histone methylationinhibitors. The epigenetic drugs may be Azacitidine (Vidaza), Decitabine(Dacogen), Vorinostat (Zolinza), Romidepsin (Istodax), or Ruxolitinib(Jakafi). For prostate cancer treatment, a preferred chemotherapeuticagent with which anti- CTLA-4 can be combined is paclitaxel (TAXOL).

In certain embodiments, the one or more additional agents are one ormore anti-glucocorticoid-induced tumor necrosis factor family receptor(GITR) agonistic antibodies. GITR is a costimulatory molecule for Tlymphocytes, modulates innate and adaptive immune system and has beenfound to participate in a variety of immune responses and inflammatoryprocesses. GITR was originally described by Nocentini et al. after beingcloned from dexamethasone-treated murine T cell hybridomas (Nocentini etal. Proc Natl Acad Sci USA 94:6216-6221 . 1997). Unlike CD28 and CTLA-4,GITR has a very low basal expression on naive CD4+ and CD8+ T cells(Ronchctti et al. Eur J Immunol 34:613-622. 2004). The observation thatGITR stimulation has immunostimulatory effects in vitro and inducedautoimmunity in vivo prompted the investigation of the antitumor potencyof triggering this pathway. A review of Modulation Of Ctla 4 And GitrFor Cancer Immunotherapy can be found in Cancer Immunology andImmunotherapy (Avogadri et al. Current Topics in Microbiology andImmunology 344. 2011). Other agents that can contribute to relief ofimmune suppression include checkpoint inhibitors targeted at anothermember of the CD28/CTLA4 Ig superfamily such as BTLA, LAG3, ICOS, PDL1or KIR (Page et a, Annual Review of Medicine 65:27 (2014)). In furtheradditional embodiments, the checkpoint inhibitor is targeted at a memberof the TNFR superfamily such as CD40, OX40, CD137, GITR, CD27 or TIM-3.In some cases targeting a checkpoint inhibitor is accomplished with aninhibitory antibody or similar molecule. In other cases, it isaccomplished with an agonist for the target; examples of this classinclude the stimulatory targets OX40 and GITR.

In certain embodiments, the one or more additional agents aresynergistic in that they increase immunogenicity after treatment. In oneembodiment the additional agent allows for lower toxicity and/or lowerdiscomfort due to lower doses of the additional therapeutic agents orany components of the combination therapy described herein. In anotherembodiment the additional agent results in longer lifespan due toincreased effectiveness of the combination therapy described herein.Chemotherapeutic treatments that enhance the immunological response in apatient have been reviewed (Zitvogel et al., Immunological aspects ofcancer chemotherapy. Nat Rev Immunol. 2008 Jan;8(1):59-73).Additionally, chemotherapeutic agents can be administered safely withimmunotherapy without inhibiting vaccine specific T-cell responses(Perez et al., A new era in anticancer peptide vaccines. Cancer May2010). In one embodiment the additional agent is administered toincrease the efficacy of the combination therapy described herein. Inone embodiment the additional agent is a chemotherapy treatment. In oneembodiment low doses of chemotherapy potentiate delayed-typehypersensitivity (DTH) responses. In one embodiment the chemotherapyagent targets regulatory T-cells. In one embodiment cyclophosphamide isthe therapeutic agent. In one embodiment cyclophosphamide isadministered prior to vaccination. In one embodiment cyclophosphamide isadministered as a single dose before vaccination (Walter et al.,Multipeptide immune response to cancer vaccine 1MA901 after single-dosecyclophosphamide associates with longer patient survival. NatureMedicine; 18:8 2012). In another embodiment, cyclophosphamide isadministered according to a metronomic program, where a daily dose isadministered for one month (Ghiringhelli et al., Metronomiccyclophosphamide regimen selectively depletes CD4+CD25+ regulatory Tcells and restores T and NK effector functions in end stage cancerpatients. Cancer Immunol Immunother 2007 56:641-648). In anotherembodiment taxanes are administered before vaccination to enhance T-celland NK-cell functions (Zitvogel et al., 2008). In another embodiment alow dose of a chemotherapeutic agent is administered with thecombination therapy described herein. In one embodiment thechemotherapeutic agent is estramustine. In one embodiment the cancer ishormone resistant prostate cancer. A ≥50% decrease in serum prostatespecific antigen (PSA) was seen in 8.7% of advanced hormone refractoryprostate cancer patients by personalized vaccination alone, whereas sucha decrease was seen in 54% of patients when the personalized vaccinationwas combined with a low dose of estramustine (Itoh et al., Personalizedpeptide vaccines: A new therapeutic modality for cancer. Cancer Sci2006; 97: 970-976). In another embodiment glucocorticoids are notadministered with or before the combination therapy described herein(Zitvogel et al., 2008). In another embodiment glucocorticoids areadministered after the combination therapy described herein. In anotherembodiment Gemcitabine is administered before, simultaneously, or afterthe combination therapy described herein to enhance the frequency oftumor specific CTL precursors (Zitvogel et al., 2008). In anotherembodiment 5-fluorouracil is administered with the combination therapydescribed herein as synergistic effects were seen with a peptide basedvaccine (Zitvogel et al., 2008). In another embodiment an inhibitor ofBraf, such as Vemurafenib, is used as an additional agent. Brafinhibition has been shown to be associated with an increase in melanomaantigen expression and T-cell infiltrate and a decrease inimmunosuppressive cytokines in tumors of treated patients (Frederick etal., BRA17 inhibition is associated with enhanced melanoma antigenexpression and a more favorable tumor microenvironment in patients withmetastatic melanoma. Clin Cancer Res. 2013; 19:1225-1231). In anotherembodiment an inhibitor of tyrosine kinases is used as an additionalagent. In one embodiment the tyrosine kinase inhibitor is used beforevaccination with the combination therapy described herein. In oneembodiment the tyrosine kinase inhibitor is used simultaneously with thecombination therapy described herein. In another embodiment the tyrosinekinase inhibitor is used to create a more immune permissive environment.In another embodiment the tyrosine kinase inhibitor is sunitinib orimatinib mesylate. It has previously been shown that favorable outcomescould be achieved with sequential administration of continuous dailydosing of sunitinib and recombinant vaccine (Farsaci et al., Consequenceof dose scheduling of sunitinib on host immune response elements andvaccine combination therapy. Int J Cancer; 130: 1948-1959). Sunitinibhas also been shown to reverse type-1 immune suppression using a dailydose of 50 mg/day (Finke et al., Sunitinib Reverses Type-1 ImmuneSuppression and Decreases T-Regulatory Cells in Renal Cell CarcinomaPatients. Clin Cancer Res 2408;14(20)). In another embodiment targetedtherapies are administered in combination with the combination therapydescribed herein. Doses of targeted therapies has been describedpreviously (Alvarez, Present and future evolution of advanced breastcancer therapy. Breast Cancer Research 2010, 12(Suppl 2):S1). In anotherembodiment temozolomide is administered with the combination therapydescribed herein. In one embodiment temozolomide is administered at 200mg/day for 5 days every fourth week of a combination therapy with thecombination therapy described herein. Results of a similar strategy havebeen shown to have low toxicity (Kyte et al., Telomerase PeptideVaccination Combined with Temozolomide: A Clinical Trial in Stage IVMelanoma Patients. Clin Cancer Res; 17(13) 2011). In another embodimentthe combination therapy is administered with an additional therapeuticagent that results in lymphopenia. In one embodiment the additionalagent is temozolomide. An immune response can still be induced underthese conditions (Sampson et al., Greater chemotherapy-inducedlymphopenia enhances tumor-specific immune responses that eliminateEGFRvIII-expressing tumor cells in patients with glioblastoma.Neuro-Oncology 13(3):324-333, 2011).

The herein-described compositions and methods may be used on patients inneed thereof with any cancer according to the general flow process shownin FIG. 2 . Patients in need thereof may receive a series of primingvaccinations with a mixture of personalized tumor-specific peptides.Additionally, over a 4 week period the priming may be followed by twoboosts during a maintenance phase. All vaccinations are subcutaneouslydelivered. The vaccine or immunogenic composition is evaluated forsafety, tolerability, immune response and clinical effect in patientsand for feasibility of producing vaccine or immunogenic composition andsuccessfully initiating vaccination within an appropriate time frame.The first cohort can consist of 5 patients, and after safety isadequately demonstrated, an additional cohort of 10 patients may beenrolled. Peripheral blood is extensively monitored for peptide-specificT-cell responses and patients are followed for up to two years to assessdisease recurrence.

Administering Combination Therapy Consistent With Standard of Care

In another aspect, the combination therapy described herein providesselecting the appropriate point to administer the combination therapy inrelation to and within the standard of care for the cancer being treatedfor a patient in need thereof. The studies described herein show thatthe combination therapy can be effectively administered even within thestandard of care that includes surgery, radiation, or chemotherapy. Thestandards of care for the most common cancers can be found on thewebsite of National Cancer Institute(http://www.cancer.gov/cancertopics). The standard of care is thecurrent treatment that is accepted by medical experts as a propertreatment for a certain type of disease and that is widely used byhealthcare professionals. Standard or care is also called best practice,standard medical care, and standard therapy. Standards of Care forcancer generally include surgery, lymph node removal, radiation,chemotherapy, targeted therapies, antibodies targeting the tumor, andimmunotherapy. Immunotherapy can include checkpoint blockers (CBP),chimeric antigen receptors (CARs), and adoptive T-cell therapy. Thecombination therapy described herein can be incorporated within thestandard of care. The combination therapy described herein may also beadministered where the standard of care has changed due to advances inmedicine.

Incorporation of the combination therapy described herein may depend ona treatment step in the standard of care that can lead to activation ofthe immune system. Treatment steps that can activate and functionsynergistically with the combination therapy have been described herein.The therapy can be advantageously administered simultaneously or after atreatment that activates the immune system.

Incorporation of the combination therapy described herein may depend ona treatment step in the standard of care that causes the immune systemto be suppressed. Such treatment steps may include irradiation, highdoses of alkylating agents and/or methotrexate, steroids such asglucosteroids, surgery, such as to remove the lymph nodes, imatinibmesylate, high doses of TNF, and taxancs (Zitvogel et al., 2008). Thecombination therapy may be administered before such steps or may beadministered after.

In one embodiment the combination therapy may be administered after bonemarrow transplants and peripheral blood stem cell transplantation. Bonemarrow transplantation and peripheral blood stem cell transplantationare procedures that restore stem cells that were destroyed by high dosesof chemotherapy and/or radiation therapy. After being treated withhigh-dose anticancer drugs and/or radiation, the patient receivesharvested stem cells, which travel to the bone marrow and begin toproduce new blood cells. A “mini-transplant” uses lower, less toxicdoses of chemotherapy and/or radiation to prepare the patient fortransplant. A “tandem transplant” involves two sequential courses ofhigh-dose chemotherapy and stem cell transplant. In autologoustransplants, patients receive their own stem cells. In syngeneictransplants, patients receive stem cells from their identical twin. Inallogeneic transplants, patients receive stem cells from their brother,sister, or parent. A person who is not related to the patient (anunrelated donor) also may be used. In some types of leukemia, thegraft-versus-tumor (GVT) effect that occurs after allogeneic BMT andPBSCT is crucial to the effectiveness of the treatment. GVT occurs whenwhite blood cells from the donor (the graft) identify the cancer cellsthat remain in the patient’s body after the chemotherapy and/orradiation therapy (the tumor) as foreign and attack them. Immunotherapywith the combination therapy described herein can take advantage of thisby vaccinating after a transplant. Additionally, the transferred cellsmay be presented with neoantigens of the combination therapy describedherein before transplantation.

In one embodiment the combination therapy is administered to a patientin need thereof with a cancer that requires surgery. In one embodimentthe combination therapy described herein is administered to a patient inneed thereof in a cancer where the standard of care is primarily surgeryfollowed by treatment to remove possible micro-metastases, such asbreast cancer. Breast cancer is commonly treated by various combinationsof surgery, radiation therapy, chemotherapy, and hormone therapy basedon the stage and grade of the cancer. Adjuvant therapy for breast canceris any treatment given after primary therapy to increase the chance oflong-term survival. Neoadjuvant therapy is treatment given beforeprimary therapy. Adjuvant therapy for breast cancer is any treatmentgiven after primary therapy to increase the chance of long-termdisease-free survival. Primary therapy is the main treatment used toreduce or eliminate the cancer. Primary therapy for breast cancerusually includes surgery, a mastectomy (removal of the breast) or alumpectomy (surgery to remove the tumor and a small amount of normaltissue around it; a type of breast-conserving surgery). During eithertype of surgery, one or more nearby lymph nodes are also removed to seeif cancer cells have spread to the lymphatic system. When a woman hasbreast-conserving surgery, primary therapy almost always includesradiation therapy. Even in early-stage breast cancer, cells may breakaway from the primary tumor and spread to other parts of the body(metastasize). Therefore, doctors give adjuvant therapy to kill anycancer cells that may have spread, even if they cannot be detected byimaging or laboratory tests.

In one embodiment the combination therapy is administered consistentwith the standard of care for Ductal carcinoma in situ (DCIS). Thestandard of care for this breast cancer type is:

-   1. Breast-conserving surgery and radiation therapy with or without    tamoxifen.-   2. Total mastectomy with or without tamoxifen.-   3. Breast-conserving surgery without radiation therapy.

The combination therapy may be administered before breast conservingsurgery or total mastectomy to shrink the tumor before surgery. Inanother embodiment the combination therapy can be administered as anadjuvant therapy to remove any remaining cancer cells.

In another embodiment patients diagnosed with stage I, II, IIIA, andOperable IIIC breast cancer are treated with the combination therapy asdescribed herein. The standard of care for this breast cancer type is:

-   1. Local-regional treatment:    -   Breast-conserving therapy (lumpectomy, breast radiation, and        surgical staging of the axilla).    -   Modified radical mastectomy (removal of the entire breast with        level I-II axillary dissection) with or without breast        reconstruction.    -   Sentinel node biopsy.-   2. Adjuvant radiation therapy postmastectomy in axillary    node-positive tumors:    -   For one to three nodes: unclear role for regional radiation        (infra/supraclavicular nodes, internal mammary nodes, axillary        nodes, and chest wall).    -   For more than four nodes or extranodal involvement: regional        radiation is advised.-   3. Adjuvant systemic therapy

In one embodiment the combination therapy is administered as aneoadjuvant therapy to shrink the tumor. In another embodiment thecombination is administered as an adjuvant systemic therapy.

In another embodiment patients diagnosed with inoperable stage IIIB orIIIC or inflammatory breast cancer are treated with the combinationtherapy as described herein. The standard of care for this breast cancertype is:

1. Multimodality therapy delivered with curative intent is the standardof care for patients with clinical stage IIIB disease.

2. Initial surgery is generally limited to biopsy to permit thedetermination of histology, estrogen-receptor (ER) andprogesterone-receptor (PR) levels, and human epidermal growth factorreceptor 2 (HER2/neu) overexpression. Initial treatment withanthracycline-based chemotherapy and/or taxane-based therapy isstandard. For patients who respond to neoadjuvant chemotherapy, localtherapy may consist of total mastectomy with axillary lymph nodedissection followed by postoperative radiation therapy to the chest walland regional lymphatics. Breast-conserving therapy can be considered inpatients with a good partial or complete response to neoadjuvantchemotherapy. Subsequent systemic therapy may consist of furtherchemotherapy. Hormone therapy should be administered to patients whosetumors are ER-positive or unknown. All patients should be consideredcandidates for clinical trials to evaluate the most appropriate fashionin which to administer the various components of multimodality regimens.

In one embodiment the combination therapy is administered as part of thevarious components of multimodality regimens. In another embodiment thecombination therapy is administered before, simultaneously with, orafter the multimodality regimens. In another embodiment the combinationtherapy is administered based on synergism between the modalities. Inanother embodiment the combination therapy is administered aftertreatment with anthracycline-based chemotherapy and/or taxane-basedtherapy (Zitvogel et al., 2008). Treatment after administering thecombination therapy may negatively affect dividing effector T-cells. Thecombination therapy may also be administered after radiation.

In another embodiment the combination therapy described herein is usedin the treatment in a cancer where the standard of care is primarily notsurgery and is primarily based on systemic treatments, such as ChronicLymphocytic Leukemia (CLL).

In another embodiment patients diagnosed with stage I, II, III, and IVChronic Lymphocytic Leukemia are treated with the combination therapy asdescribed herein. The standard of care for this cancer type is:

-   1. Observation in asymptomatic or minimally affected patients-   2. Rituximab-   3. Ofatumomab-   4. Oral alkylating agents with or without corticosteroids-   5. Fludarabine, 2-chlorodeoxyadenosine, or pentostatin-   6. Bendamustine-   7. Lenalidomide-   8. Combination chemotherapy.    -   combination chemotherapy regimens include the following:        -   Fludarabine plus cyclophosphamide plus rituximab.        -   Fludarabine plus rituximab as seen in the CLB-9712 and            CLB-9011 trials.        -   Fludarabine plus cyclophosphamide versus fludarabine plus            cyclophosphamide plus rituximab.        -   Pentostatin plus cyclophosphamide plus rituximab as seen in            the MAYO-MC0183 trial, for example.        -   Ofatumumab plus fludarabine plus cyclophosphamide.        -   CVP: cyclophosphamide plus vincristine plus prednisone.        -   CHOP: cyclophosphamide plus doxorubicin plus vincristine            plus prednisone.        -   Fludarabine plus cyclophosphamide versus fludarabine as seen            in the E2997 trial [NCT00003764] and the LRF-CLL4 trial, for            example.        -   Fludarabine plus chlorambucil as seen in the CLB-9011 trial,            for example.-   9. Involved-field radiation therapy.-   10. Alemtuzumab-   11. Bone marrow and peripheral stem cell transplantations are under    clinical evaluation.-   12. Ibrutinib

In one embodiment the combination therapy is administered before,simultaneously with or after treatment with Rituximab or Ofatumomab. Asthese are monoclonal antibodies that target B-cells, treatment with thecombination therapy may be synergistic. In another embodiment thecombination therapy is administered after treatment with oral alkylatingagents with or without corticosteroids, and Fludarabine,2-chlorodeoxyadenosine, or pentostatin, as these treatments maynegatively affect the immune system if administered before. In oneembodiment bendamustine is administered with the combination therapy inlow doses based on the results for prostate cancer described herein. Inone embodiment the combination therapy is administered after treatmentwith bendamustine.

Vaccine or Immunogenic Composition Kits and Co-Packaging

In an aspect, the invention provides kits containing any one or more ofthe elements discussed herein to allow administration of the combinationtherapy. Elements may be provided individually or in combinations, andmay be provided in any suitable container, such as a vial, a bottle, ora tube. In some embodiments, the kit includes instructions in one ormore languages, for example in more than one language. In someembodiments, a kit comprises one or more reagents for use in a processutilizing one or more of the elements described herein. Reagents may beprovided in any suitable container. For example, a kit may provide oneor more delivery or storage buffers. Reagents may be provided in a formthat is usable in a particular process, or in a form that requiresaddition of one or more other components before use (e.g. in concentrateor lyophilized form). A buffer can be any buffer, including but notlimited to a sodium carbonate buffer, a sodium bicarbonate buffer, aborate buffer, a Tris buffer, a MOPS buffer, a HEPES buffer, andcombinations thereof. In some embodiments, the buffer is alkaline. Insome embodiments, the buffer has a pH from about 7 to about 10. In someembodiments, the kit comprises one or more of the vectors, proteinsand/or one or more of the polynucleotides described herein. The kit mayadvantageously allow the provision of all elements of the systems of theinvention. Kits can involve vector(s) and/or particle(s) and/ornanoparticle(s) containing or encoding RNA(s) for 1-50 or moreneoantigen mutations to be administered to an animal, mammal, primate,rodent, etc., with such a kit including instructions for administeringto such a eukaryote; and such a kit can optionally include any of theanti-cancer agents described herein. The kit may include any of thecomponents above (e.g. vector(s) and/or particlc(s) and/ornanoparticle(s) containing or encoding RNA(s) for 1-50 or moreneoantigen mutations, neoantigen proteins or peptides, checkpointinhibitors) as well as instructions for use with any of the methods ofthe present invention.

In one embodiment the kit contains at least one vial with an immunogeniccomposition or vaccine and at least one vial with an anticancer agent.In one embodiment kits may comprise ready to use components that aremixed and ready to administer. In one aspect a kit contains a ready touse immunogenic or vaccine composition and a ready to use anti-canceragent. The ready to use immunogenic or vaccine composition may compriseseparate vials containing different pools of immunogenic compositions.The immunogenic compositions may comprise one vial containing a viralvector or DNA plasmid and the other vial may comprise immunogenicprotein. The ready to use anticancer agent may comprise a cocktail ofanticancer agents or a single anticancer agent. Separate vials maycontain different anti-cancer agents. In another embodiment a kit maycontain a ready to use anti-cancer agent and an immunogenic compositionor vaccine in a ready to be reconstituted form. The immunogenic orvaccine composition may be freeze dried or lyophilized. The kit maycomprise a separate vial with a reconstitution buffer that can be addedto the lyophilized composition so that it is ready to be administered.The buffer may advantageously comprise an adjuvant or emulsion accordingto the present invention. In another embodiment the kit may comprise aready to reconstitute anti-cancer agent and a ready to reconstituteimmunogenic composition or vaccine. In this aspect both may belyophilized. In this aspect separate reconstitution buffers for each maybe included in the kit. The buffer may advantageously comprise anadjuvant or emulsion according to the present invention. In anotherembodiment the kit may comprise single vials containing a dose ofimmunogenic composition and anti-cancer agent that are administeredtogether. In another aspect multiple vials are included so that one vialis administered according to a treatment timeline. One vial may onlycontain the anti-cancer agent for one dose of treatment, another maycontain both the anti-cancer agent and immunogenic composition foranother dose of treatment, and one vial may only contain the immunogeniccomposition for yet another dose. In a further aspect the vials arelabeled for their proper administration to a patient in need thereof.The immunogen or anti-cancer agents of any embodiment may be in alyophilized form, a dried form or in aqueous solution as describedherein. The immunogen may be a live attenuated virus, protein, ornucleic acid as described herein.

In one embodiment the anticancer agent is one that enhances the immunesystem to enhance the effectiveness of the immunogenic composition orvaccine. In a preferred embodiment the anti-cancer agent is a checkpointinhibitor. In another embodiment the kit contains multiple vials ofimmunogenic compositions and anti-cancer agents to be administered atdifferent time intervals along a treatment plan. In another embodimentthe kit may comprise separate vials for an immunogenic composition foruse in priming an immune response and another immunogenic composition tobe used for boosting. In one aspect the priming immunogenic compositioncould be DNA or a viral vector and the boosting immunogenic compositionmay be protein. Either composition may be lyophilized or ready foradministering. In another embodiment different cocktails of anti-canceragents containing at least one anti-cancer agent are included indifferent vials for administration in a treatment plan.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined in the appended claims.

The present invention is further illustrated in the following Exampleswhich are given for illustration purposes only and are not intended tolimit the invention in any way.

EXAMPLES Example 1 Cancer Vaccine Testing Protocol

The herein-described compositions and methods may be tested on 15patients with high-risk melanoma (fully resected stages IIIB, IIIC andIVM1a,b) according to the general flow process shown in FIG. 2 .Patients may receive a series of priming vaccinations with a mixture ofpersonalized tumor-specific peptides and poly-ICLC over a 4 week periodfollowed by two boosts during a maintenance phase. All vaccinations aresubcutaneously delivered. The vaccine or immunogenic composition isevaluated for safety, tolerability, immune response and clinical effectin patients and for feasibility of producing vaccine or immunogeniccomposition and successfully initiating vaccination within anappropriate time frame. The first cohort can consist of 5 patients, andafter safety is adequately demonstrated, an additional cohort of 10patients may be enrolled. Peripheral blood is extensively monitored forpeptide-specific T-cell responses and patients are followed for up totwo years to assess disease recurrence.

As described herein, there is a large body of evidence in both animalsand humans that mutated epitopes are effective in inducing an immuneresponse and that cases of spontaneous tumor regression or long termsurvival correlate with CD8+ T-cell responses to mutated epitopes(Buckwalter and Srivastava PK. “It is the antigen(s), stupid” and otherlessons from over a decade of vaccitherapy of human cancer. Seminars inimmunology 20:296-300 (2008); Karanikas et al, High frequency ofcytolytic T lymphocytes directed against a tumor-specific mutatedantigen detectable with HLA tetramers in the blood of a lung carcinomapatient with long survival. Cancer Res. 61:3718-3724 (2001); Lennerz etal, The response of autologous T cells to a human melanoma is dominatedby mutated neoantigens. Proc Natl Acad Sci U S A.102:16013 (2005)) andthat “immunoediting” can be tracked to alterations in expression ofdominant mutated antigens in mice and man (Matsushita et al, Cancerexome analysis reveals a T-cell-dependent mechanism of cancerimmunoediting Nature 482:400 (2012); DuPage et al, Expression oftumor-specific antigens underlies cancer immunoediting Nature 482:405(2012); and Sampson et al, Immunologic escape after prolongedprogression-free survival with epidermal growth factor receptor variantIII peptide vaccination in patients with newly diagnosed glioblastoma JClin Oncol. 28:4722-4729 (2010)).

Next-generation sequencing can now rapidly reveal the presence ofdiscrete mutations such as coding mutations in individual tumors, mostcommonly single amino acid changes (e.g., missense mutations) and lessfrequently novel stretches of amino acids generated by frame-shiftinsertions/deletions/gene fusions, read-through mutations in stopcodons, and translation of improperly spliced introns (e.g., neoORFs).NeoORFs are particularly valuable as immunogens because the entirety oftheir sequence is completely novel to the immune system and so areanalogous to a viral or bacterial foreign antigen. Thus, neoORFs: (1)are highly specific to the tumor (i.e. there is no expression in anynormal cells); (2) can bypass central tolerance, thereby increasing theprecursor frequency of neoantigen-specific CTLs. For example, the powerof utilizing analogous foreign sequences in a therapeutic anti-cancervaccine was recently demonstrated with peptides derived from humanpapilloma virus (HPV). ~50% of the 19 patients with pre-neoplastic,viral-induced disease who received 3-4 vaccinations of a mix of HPVpeptides derived from the viral oncogenes E6 and E7 maintained acomplete response for ≥24 months (Kenter et a, Vaccination againstHPV-16 Oncoproteins for Vulvar Intraepithelial Neoplasia NEJM 361:1838(2009)).

Sequencing technology has revealed that each tumor contains multiple,patient-specific mutations that alter the protein coding content of agene. Such mutations create altered proteins, ranging from single aminoacid changes (caused by missense mutations) to addition of long regionsof novel amino acid sequence due to frame shifts, read-through oftermination codons or translation of intron regions (novel open readingframe mutations; neoORFs). These mutated proteins are valuable targetsfor the host’s immune response to the tumor as, unlike native proteins,they are not subject to the immune-dampening effects of self-tolerance.Therefore, mutated proteins are more likely to be immunogenic and arealso more specific for the tumor cells compared to normal cells of thepatient.

Utilizing recently improved algorithms for predicting which missensemutations create strong binding peptides to the patient’s cognate MHCmolecules, a set of peptides representative of optimal mutated epitopes(both neoORF and missense) for each patient is identified andprioritized and up to 20 or more peptides are prepared for immunization(Zhang et al, Machine learning competition in immunology - Prediction ofHLA class I binding peptides J Immunol Methods 374:1 (2011); Lundegaardet al Prediction of epitopes using neural network based methods JImmunol Methods 374:26 (2011)). Peptides ~20-35 amino acids in length issynthesized because such “long” peptides undergo efficientinternalization, processing and cross-presentation in professionalantigen-presenting cells such as dendritic cells, and have been shown toinduce CTLs in humans (Melief and van der Burg, Immunotherapy ofestablished (pre) malignant disease by synthetic long peptide vaccinesNature Rev Cancer 8:351 (2008)).

In addition to a powerful and specific immunogen, an effective immuneresponse advantageously includes a strong adjuvant to activate theimmune system (Speiser and Romero, Molecularly defined vaccines forcancer immunotherapy, and protective T cell immunity Seminars in Immunol22: 144 (2010)). For example, Toll-like receptors (TLRs) have emerged aspowerful sensors of microbial and viral pathogen “danger signals”,effectively inducing the innate immune system, and in turn, the adaptiveimmune system (Bhardwaj and Gnjatic, TLR AGONISTS: Are They GoodAdjuvants? Cancer J. 16:382-391 (2010)). Among the TLR agonists,poly-ICLC (a synthetic double-stranded RNA mimic) is one of the mostpotent activators of myeloid-derived dendritic cells. In a humanvolunteer study, poly-ICLC has been shown to be safe and to induce agene expression profile in peripheral blood cells comparable to thatinduced by one of the most potent live attenuated viral vaccines, theyellow fever vaccine YF-17D (Caskey et al, Synthetic double-stranded RNAinduces innate immune responses similar to a live viral vaccine inhumans J Exp Med 208:2357 (2011)). Hiltonol®, a GMP preparation ofpoly-ICLC prepared by Oncovir, Inc, is utilized as the adjuvant.

Example 2 Target Patient Population

Patients with stage IIIB, IIIC and IVM1a,b, melanoma have a significantrisk of disease recurrence and death, even with complete surgicalresection of disease (Balch et al, Final Version of 2009 AJCC MelanomaStaging and Classification J Clin Oncol 27:6199 - 6206 (2009)). Anavailable systemic adjuvant therapy for this patient population isinterferon-α (IFNα) which provides a measurable but marginal benefit andis associated with significant, frequently dose-limiting toxicity(Kirkwood et al, Interferon alfa-2b Adjuvant Therapy of High-RiskResected Cutaneous Melanoma: The Eastern Cooperative Oncology GroupTrial EST 1684 J Clin Oncol 14:7-17 (1996); Kirkwood et al, High- andLow-dose Interferon Alpha-2b in High-Risk Melanoma: First Analysis ofIntergroup Trial E1690/S9111/C9190 J Clin Oncol 18:2444 -2458 (2000)).These patients are not immuno-compromised by previous cancer-directedtherapy or by active cancer and thus represent an excellent patientpopulation in which to assess the safety and immunological impact of thevaccine. Finally, current standard of care for these patients docs notmandate any treatment following surgery, thus allowing for the 8 - 10week window for vaccine preparation.

The target population is cutaneous melanoma patients with clinicallydetectable, histologically confirmed nodal (local or distant) or intransit metastasis, who have been fully resected and are free of disease(most of stage IIIB (because of the need to have adequate tumor tissuefor sequencing and cell line development, patients with ulceratedprimary tumor but micrometastatic lymph nodes (T1-4b, N1a or N2a) isexcluded.), all of stage IIIC, and stage IVM1a, b). These may bepatients at first diagnosis or at disease recurrence after previousdiagnosis of an earlier stage melanoma.

Tumor harvest: Patients can undergo complete resection of their primarymelanoma (if not already removed) and all regional metastatic diseasewith the intent of rendering them free of melanoma. After adequate tumorfor pathological assessment has been harvested, remaining tumor tissueis placed in sterile media in a sterile container and prepared fordisaggregation. Portions of the tumor tissue is used for whole-exome andtranscriptome sequencing and cell line generation and any remainingtumor is frozen.

Normal tissue harvest: A normal tissue sample (blood or sputum sample)is taken for whole exome sequencing.

Patients with clinically evident locoregional metastatic disease orfully resectable distant nodal, cutaneous or lung metastatic disease(but absence of unresectable distant or visceral metastatic disease) isidentified and enrolled on the study. Entry of patients prior to surgeryis necessary in order to acquire fresh tumor tissue for melanoma cellline development (to generate target cells for in vitro cytotoxicityassays as part of the immune monitoring plan).

Example 3 Dose and Schedule

For patients who have met all pre-treatment criteria, vaccineadministration can commence as soon as possible after the study drug hasarrived and has met incoming specifications. For each patient, there isfour separate study drugs, each containing 5 of 20 patient-specificpeptides. Immunizations may generally proceed according to the scheduleshown in FIG. 3 .

Patients are treated in an outpatient clinic. Immunization on eachtreatment day can consist of four 1 ml subcutaneous injections, eachinto a separate extremity in order to target different regions of thelymphatic system to reduce antigenic competition. If the patient hasundergone complete axillary or inguinal lymph node dissection, vaccinesare administered into the right or left midriff as an alternative. Eachinjection can consist of 1 of the 4 study drugs for that patient and thesame study drug is injected into the same extremity for each cycle. Thecomposition of each 1 ml injection is:

-   0.75 ml study drug containing 300 µg each of 5 patient-specific    peptides-   0.25 ml (0.5 mg) of 2 mg/ml poly-ICLC (Hiltonol®)

During the induction/priming phase, patients are immunized on days 1, 4,8, 15 and 22. In the maintenance phase, patients can receive boosterdoses at weeks 12 and 24.

Blood samples may be obtained at multiple time points: pre- (baseline;two samples on different days); day 15 during priming vaccination; fourweeks after the induction/priming vaccination (week 8); pre- (week 12)and post- (week 16) first boost; pre- (week 24) and post-(week 28)second boost 50 - 150 ml blood is collected for each sample (except week16). The primary immunological endpoint is at week 16, and hencepatients can undergo leukapheresis (unless otherwise indicated based onpatient and physician assessment).

Example 4 Immune Monitoring

The immunization strategy is a “prime-boost” approach, involving aninitial series of closely spaced immunizations to induce an immuneresponse followed by a period of rest to allow memory T-cells to beestablished. This is followed by a booster immunization, and the T-cellresponse 4 weeks after this boost is expected to generate the strongestresponse and is the primary immunological endpoint. Global immunologicalresponse is initially monitored using peripheral blood mononuclear cellsfrom this time point in an 18 hr ex vivo ELISPOT assay, stimulating witha pool of overlapping 15mer peptides (11 aa overlap) comprising all theimmunizing epitopes. Pre-vaccination samples are evaluated to establishthe baseline response to this peptide pool. As warranted, additionalPBMC samples are evaluated to examine the kinetics of the immuneresponse to the total peptide mix. For patients demonstrating responsessignificantly above baseline, the pool of all 15mers are de-convolutedto determine which particular immunizing peptide(s) were immunogenic. Inaddition, a number of additional assays are conducted on a case-by-casebasis for appropriate samples:

-   The entire 15mer pool or sub-pools are used as stimulating peptides    for intracellular cytokine staining assays to identify and quantify    antigen-specific CD4+, CD8+, central memory and effector memory    populations-   Similarly, these pools are used to evaluate the pattern of cytokines    secreted by these cells to determine the TH1 vs TH2 phenotype-   Extracellular cytokine staining and flow cytometry of unstimulated    cells are used to quantify Treg and myeloid-derived suppressor cells    (MDSC).-   If a melanoma cell line is successfully established from a    responding patient and the activating epitope can be identified,    T-cell cytotoxicity assays are conducted using the mutant and    corresponding wild type peptide-   PBMC from the primary immunological endpoint is evaluated for    “epitope spreading” by using known melanoma tumor associated    antigens as stimulants and by using several additional identified    mutated epitopes that were not selected to be among the immunogens,    as shown in FIG. 4 .

Immuno-histochemistry of the tumor sample is conducted to quantify CD4+,CD8+, MDSC, and Treg infiltrating populations.

Example 5 Clinical Efficacy in Patients With Metastatic Disease

Vaccine treatment of patients with metastatic disease is complicated bytheir need for an effective therapy for the active cancer and theconsequent absence of an off treatment time window for vaccinepreparation. Furthermore, these cancer treatments may compromise thepatient’s immune system, possibly impeding the induction of an immuneresponse. With these considerations in mind, settings may be chosenwhere timing of vaccine preparation fits temporally with other standardcare approaches for the particular patient population and/or where suchstandard care is demonstrably compatible with an immunotherapeuticapproach. There are two types of settings that may be pursued:

-   1. Combination with checkpoint blockade: Checkpoint blockade    antibodies have emerged as an effective immunotherapy for metastatic    melanoma (Hodi et al, Improved Survival with Ipilimumab in Patients    with Metastatic Melanoma NEJM 363:711 - 723 (2010)) and are being    actively pursued in other disease settings including non-small cell    lung cancer (NSCLC) and renal cell carcinoma (Topalian et al,    Safety, Activity, and Immune Correlates of Anti-PD-1 Antibody in    Cancer NEJM 366:2443-2454 (2012); Brahmer et al, Safety and Activity    of Anti-PD-L1 Antibody in Patients with Advanced Cancer NEJM    366:2455-2465(2012)). Although the mechanism of action is not    proven, both reversal of relief from local immunosuppression and    enhancement of an immune response are possible explanations.    Integrating a powerful vaccine to initiate an immune response with    checkpoint blockade antibodies may provide synergies, as observed in    multiple animal studies (van Elsas et al Combination immunotherapy    of B16 melanoma using anti-cytotoxic T lymphocyte-associated antigen    4 (CTLA-4)and granulocyte/macrophage colony-stimulating factor    (GM-CSF)-producing vaccines induces rejection of subcutaneous and    metastatic tumors accompanied by autoimmune depigmentation J Exp Med    190:35- 366 (1999); Li et al, Anti-programmed death-1 synergizes    with granulocyte macrophage colony-stimulating factor -secreting    tumor cell immunotherapy providing therapeutic benefit to mice with    established tumors Clin Cancer Res 15:1623 - 1634 (2009);    Pardoll, D. M. The blockade of immune checkpoints in cancer    immunotherapy Nature Reviews Cancer 12:252 - 264 (2012); Curran et    al. PD-1 and CTLA-4 combination blockade expands infiltrating T    cells and reduces regulatory T and myeloid cells within B16 melanoma    tumors. Proc Natl Acad Sci U S A. 2010 Mar 2;107(9):4275-80; Curran    et al. Tumor vaccines expressing flt3 ligand synergize with ctla-4    blockade to reject preimplanted tumors. Cancer Res. 2009 Oct    1;69(19):7747-55). Patients can be immediately started on checkpoint    blockade therapy while vaccine is being prepared and once prepared,    the vaccine dosing can be integrated with antibody therapy, as    illustrated in FIG. 5 ; and-   2. Combination with standard treatment regimens exhibiting    beneficial immune properties.    -   a) Renal cell carcinoma (RCC) patients who present with        metastatic disease typically undergo surgical de-bulking        followed by systemic treatment, which is commonly with one of        the approved tyrosine kinase inhibitors (TKI) such as sunitinib,        pazopanib and sorafenib. Of the approved TKIs, sunitinib has        been shown to increase TH1 responsiveness and decrease Treg and        myeloid-derived suppressor cells (Finke et al, Sunitinib        reverses Type-1 immune suppression and decreases T-regulatory        cells in renal cell carcinoma patients Clin Can Res 14:6674 -        6682 (2008); Terme et al, VEGFA-VEGFR pathway blockade inhibits        tumor-induced regulatory T cell proliferation in colorectal        cancer (Cancer Research Author Manuscript published Online        (2102)). The ability to immediately treat patients with an        approved therapy that does not compromise the immune system        provides the needed window to prepare the vaccine and could        provide synergy with a vaccine therapy. In addition,        cyclophosphamide (CTX) has been implicated in multiple animal        and human studies to have an inhibitory effect on Treg cells and        a single dose of CTX prior to a vaccine has been recently shown        to improve survival in RCC patients who responded to the vaccine        (Walter et al, Multipeptide immune response to a cancer vaccine        IMA901 after single-dose cyclophosphamide associates with longer        patient survival Nature Medicine 18:1254- 1260 (2012)). Both of        these immune-synergistic approaches have been utilized in a        recently completed phase 3 study of a native peptide vaccine in        RCC (ClinicalTrials.gov, NCT01265901 IMA901 in Patients        Receiving Sunitinib for Advanced/Metastatic Renal Cell        Carcinoma);    -   b) Alternatively, standard treatment of glioblastoma (GBM)        involves surgery, recovery and follow-up radiation and low dose        temozolomide (TMZ) followed by a four week rest period before        initiating standard dose TMZ. This standard treatment provides a        window for vaccine preparation followed by initiation of        vaccination prior to starting standard dose TMZ. Interestingly,        in a study in metastatic melanoma, peptide vaccination during        standard dose TMZ treatment increased the measured immune        responsiveness compared to vaccination alone, suggesting        additional synergistic benefit (Kyte et al, Telomerase peptide        vaccination combined with temozolomide: a clinical trial in        stage IV melanoma patients Clin Cancer Res 17:4568 (2011)).

Example 6 Neoantigen Preparation

Following surgical resection of the tumor, a portion of the tumor tissueand a blood sample is transferred immediately to the facility where itis assigned a unique identification code for further tracking. The tumortissue is disaggregated with collagenase and separate portions arefrozen for nucleic acid (DNA and RNA) extraction. The blood sample isimmediately transferred to a facility for nucleic acid extraction. DNAand/or RNA extracted from the tumor tissue is used for whole-exomesequencing (e.g., by using the Illumina HiSeq platform) and to determineHLA typing information. It is contemplated within the scope of theinvention that missense or neoORF neoantigenic peptides may be directlyidentified by protein-based techniques (e.g., mass spectrometry).

Bioinformatics analysis are conducted as follows. Sequence analysis ofthe Exome and RNA - SEQ fast Q files leverage existing bioinformaticpipelines that have been used and validated extensively in large-scaleprojects such as the TCGA for many patient samples (e.g., Chapman et al,2011, Stransky et al, 2011, Berger et al, 2012). There are twosequential categories of analyses: data processing and cancer genomeanalysis.

Data processing pipeline: The Picard data processing pipeline(picard.sourceforge.net/) was developed by the Sequencing Platform. Rawdata extracted from (e.g., Illumina) sequencers for each tumor andnormal sample is subjected to the following processes using variousmodules in the Picard pipeline:

(i) Data conversion: Raw Illumina data is converted to the standard BAMformat and basic QC metrics pertaining to the distribution of basesexceeding different quality thresholds are generated.

(ii) Alignment: The Burrows-Wheeler Alignment Tool (BWA) is used toalign read pairs to the human genome (hg19).

(iii) Mark Duplicates: PCR and optical duplicates are identified basedon read pair mapping positions and marked in the final BAM file.

(iv) Indel Realignment: Reads that align to known insertion and deletionpolymorphic sites in the genome is examined and those sites where thelog odds (LOD) score for improvement upon realignment is at least 0.4 iscorrected.

(v) Quality Recalibration: Original base quality scores reported by theIllumina pipeline is recalibrated based on the read-cycle, the lane, theflow cell tile, the base in question and the preceding base. Therecalibration assumes that all mismatches in non-dbSNP positions are dueto errors which enable recalibration of the probability of error in eachcategory of interest as the fraction of mismatches amongst the totalnumber of observations.

(vi) Quality Control: The final BAM file is processed to generateextensive QC metrics including read quality by cycle, distribution ofquality scores, summary of alignment and the insert size distribution.Data that fails quality QC is blacklisted.

(vii) Identity Verification: Orthogonally collected sample genotype dataat -100 known SNP positions are checked against the sequence data toconfirm the identity of the sample. A LOD score of ≥ 10 is used as athreshold for confirmation of identity. Data that fails identity QC isblacklisted.

(viii) Data Aggregation: All data from the same sample is merged and themark duplicates step is repeated. Novel target regions containingputative short insertions and deletion regions are identified and theindel realignment step is performed at these loci.

(ix) Local realignment around putative indcls in aggregated data: Noveltarget regions containing putative short insertions and deletions areidentified and a local realignment step is performed at these loci(e.g., using the GATK RealignerTargetCreator and IndelRealigner modules)to ensure consistency and correctness of indel calls.

(x) Quality Control on Aggregated Data: QC metrics such as alignmentsummary and insert size distribution is recomputed. Additionally a setof metrics that evaluate the rate of oxidative damage in the early stepsof the library constructions process caused by acoustic shearing of DNAin the presence of reactive contaminants from the extraction process aregenerated.

The output of Picard is a bam file (Li et al, 2009) (see, e.g.,http://samtools.sourceforge.net/SAM1.pdf) that stores the basesequences, quality scores, and alignment details for all reads for thegiven sample.

Cancer Mutation Detection Pipeline: Tumor and matched normal bam filesfrom the Picard pipeline is analyzed as described herein:

-   1. Quality Control    -   (i). The Capseg program is applied to tumor and matched normal        exome samples to get the copy number profiles. The CopyNumberQC        tool can then be used to manually inspect the generated profiles        and assess tumor/normal sample mix-ups. Normal samples that have        noisy profiles as well as cases where the tumor sample has lower        copy number variation than the corresponding normal is flagged        and tracked through the data generation and analysis pipelines        to check for mix-ups.    -   (ii). Tumor purity and ploidy is estimated by the ABSOLUTE tool        15 based on Capseg-generated copy number profiles. Very noisy        profiles might result from sequencing of highly degraded        samples. No tumor purity and ploidy estimates would be possible        in such cases and the corresponding sample is flagged.    -   (iii). ContEst (Cibulskis et al, 2011) is used to determine the        level of cross-sample contamination in samples. Samples with        greater than 4% contamination is discarded.-   2. Identification of somatic single nucleotide variations (SSNVs)    -   Somatic base pair substitutions are identified by analyzing        tumor and matched normal bams from a patient using a Bayesian        statistical framework called muTect (Cibulskis et al, 2013). In        the preprocessing step, reads with a preponderance of low        quality bases or mismatches to the genome are filtered out.        Mutect then computes two log-odds (LOD) scores which encapsulate        confidence in presence and absence of the variant in the tumor        and normal samples respectively. In the post-processing stage        candidate mutations are filtered by six filters to account for        artifacts of capture, sequencing and alignment:        -   (i) Proximal gap: removes false positives that arise due to            the presence of misaligned indels in the vicinity of the            event. Samples with ≥ 3 reads with insertions or deletions            in a 11-bp window around the candidate mutation are            rejected.        -   (ii) Poor mapping: discards false positives that arise by            virtue of ambiguous placement of reads in the genome.            Rejects candidates if ≥ 50% reads in tumor and normal            samples have mapping quality zero or if there are no reads            harboring the mutant allele with mapping quality ≥ 20.        -   (iii) Trialleleic sites: discards sites that are            heterozygous in the normal since these have a tendency to            generate many false positives.        -   (iv) Strand bias: removes false positives caused by            context-specific sequencing errors where a large fraction of            reads harboring the mutation have the same orientation.            Rejects candidates where the strand-specific LOD is < 2            where the sensitivity to pass that threshold is ≥ 90%.        -   (v) Clustered position: rejects false positives due to            alignment errors characterized by the alternative allele            occurring at a fixed distance from the start or end of the            read alignment. Rejects if the median distance from the            start and end of the reads are ≤ 10 which implies that the            mutation is at the start or end of the alignment, or if the            median absolute deviation of the distances are ≤ 3 which            implies that the mutations are clustered.        -   (vi) Observed in control: discards false positives in the            tumor where there is evidence of occurrence of the alternate            allele in the normal sample beyond what is expected by            random sequencing errors. Rejects if there are ≥ 2 reads            containing the alternate allele in the normal sample or if            they are in ≥ 3% of the reads, and if the sum of their            quality scores are > 20.    -   In addition to these 6 filters, candidates are compared against        a panel of normal samples and those that are found to be present        as germline variants in two or more normal samples are rejected.        The final set of mutations can then be annotated with the        Oncotator tool by several fields including genomic region,        codon, cDNA and protein changes.-   3. Identification of somatic small insertions and deletions    -   The local realignment output described herein (see “Local        realignment around putative indels in aggregated data”, supra)        is used to predict candidate somatic and germline indels based        on assessment of reads supporting the variant exclusively in        tumor or both in tumor and normal bams respectively. Further        filtering based on number and distribution of mismatches and        base quality scores are done (McKenna et al, 2010, DePristo et        al, 2011). All indels are manually inspected using the        Integrated Genomics Viewer (Robinson et al, 2011)        (www.broadinstitute.org/igv) to ensure high-fidelity calls.-   4. Gene fusion detection    -   The first step in the gene fusion detection pipeline is        alignment of tumor RNA-Seq reads to a library of known gene        sequences following by mapping of this alignment to genomic        coordinates. The genomic mapping helps collapse multiple read        pairs that map to different transcript variants that share exons        to common genomic locations. The DNA aligned bam file is queried        for read pairs where the two mates map to two different coding        regions that are either on different chromosomes or at least 1        MB apart if on the same chromosome. It can also be required that        the pair ends aligned in their respective genes be in the        direction consistent with coding-->coding 5′-> 3′ direction of        the (putative) fusion mRNA transcript. A list of gene pairs        where there are at least two such ‘chimeric’ read pairs are        enumerated as the initial putative event list subject to further        refinement. Next, all unaligned reads are extracted from the        original bam file, with the additional constraint that their        mates were originally aligned and map into one of the genes in        the gene pairs obtained as described herein. An attempt can then        be made to align all such originally unaligned reads to the        custom “reference” built of all possible exon-exon junctions        (full length, boundary-to-boundary, in coding 5′-> 3′ direction)        between the discovered gene pairs. If one such originally        unaligned read maps (uniquely) onto a junction between an exon        of gene X and an exon of gene Y, and its mate was indeed mapped        to one of the genes X or Y, then such a read is marked as a        “fusion” read. Gene fusion events are called in cases where        there is at least one fusion read in correct relative        orientation to its mate, without excessive number of mismatches        around the exon:exon junction and with a coverage of at least 10        bp in either gene. Gene fusions between highly homologous genes        (ex. HLA family) are likely spurious and is filtered out.-   5. Estimation of clonality    -   Bioinformatic analysis may be used to estimate clonality of        mutations. For example, the ABSOLUTE algorithm (Carter et al,        2012, Landau et al, 2013) may be used to estimate tumor purity,        ploidy, absolute copy numbers and clonality of mutations.        Probability density distributions of allelic fractions of each        mutation is generated followed by conversion to cancer cell        fractions (CCFs) of the mutations. Mutations are classified as        clonal or subclonal based on whether the posterior probability        of their CCF exceeds 0.95 is greater or lesser than 0.5        respectively.-   6. Quantification of expression    -   The TopHat suite (Langmead et al, 2009) is used to align RNA-Seq        reads for the tumor and matched normal bams to the hg19 genome.        The quality of RNA-Seq data is assessed by the RNA-SeQC (DeLuca        et al., 2012) package. The RSEM tool (Li et al., 2011) can then        be used to estimate gene and isoform expression levels. The        generated reads per kilobase per million and tau estimates are        used to prioritize neoantigens identified in each patient as        described elsewhere.-   7. Validation of mutations in RNA-Seq-   8. Confirmation of the somatic mutations identified by analysis of    whole exome data as described herein (including single nucleotide    variations, small insertions and deletions and gene fusions) are    assessed by examining the corresponding RNA-Seq tumor BAM file of    the patient. For each variant locus, a power calculation based on    the beta-binomial distribution is performed to ensure that there is    at least 95% power to detect it in the RNA-Scq data. A capture    identified mutation is considered validated if there are at least 2    reads harboring the mutation for adequately powered sites.

Selection of Tumor-Specific Mutation-Containing Epitopes: All missensemutations and neoORFs are analyzed for the presence ofmutation-containing epitopes using the neuralnetwork based algorithmnetMHC provided and maintained by the Center for Biological SequenceAnalysis, Technical University of Denmark, Netherlands. This family ofalgorithms were rated the top epitope prediction algorithms based on acompetition recently completed among a series of related approaches(ref). The algorithms were trained using an artificial neural networkbased approach on 69 different human HLA A and B alleles covering 99% ofthe HLA-A alleles and 87% of the HLA-B alleles found in the Caucasianpopulation, the major ethnic group in the target patient population inthe local area. The most up-do-date version is utilized (v2.4).

The accuracy of the algorithms were evaluated by conducting predictionsfrom mutations found in CLL patients for whom the HLA allotypes wereknown. The included allotypes were A0101, A0201, A0310, A1101, A2402,A6801, B0702, B0801, B1501. Predictions were made for all 9mcr and 10mer peptides spanning each mutation using netMHCpan in mid-2011. Basedon these predictions, seventy-four (74) 9mer peptides and sixty-three(63) 10mer peptides, most with predicted affinities below 500 nM, weresynthesized and the binding affinity was measured using a competitivebinding assay (Sette).

The predictions for these peptides were repeated in March 2013 usingeach of the most up to date versions of the netMHC.’ servers (netMHCpan,netMHC and netMHCcons). These three algorithms were the top ratedalgorithms among a group of 20 used in a competition in 2012 (Zhang etal). The observed binding affinities were then evaluated with respect toeach of the new predictions. For each set of predicted and observedvalues, the % of correct predictions for each range is given, as well asthe number of samples. The definition for each range is as follows:

-   0 - 150: Predicted to have an affinity equal to or lower than 150 nM    and measured to have an affinity equal to or lower than 150 nM.-   0 - 150*: Predicted to have an affinity equal to or lower than 150    nM and measured to have an affinity equal to or lower than 500 nM.-   151 - 500 nM: Predicted to have an affinity greater than 150 nM but    equal to or lower than 500 nM and measured to have an affinity equal    to or below 500 nM.-   FN (> 500 nM): False Negatives --- Predicted to have an affinity    greater than 500 nM but measured to have an affinity equal to or    below 500 nM.

For 9mer peptides (Table 1) , there was little difference between thealgorithms, with the slightly higher value for the 151 - 500 nM rangefor netMHC cons not judged to be significant because of the low numberof samples.

TABLE 1 Range (nM) 9mer PAN 9mer netMHC 9mer CONS 0-150 76% (33) 78%(37) 76% (34) 0-150* 91% (33) 89% (37) 88% (34) 151-500 50% (28) 50%(14) 62% (13) FN (>500) 38% (13) 39% (23) 41% (27)

For 10mer peptides (Table 2), again there was little difference betweenthe algorithms except that netMHC produced significantly more falsepositives than netMHCpan or netMMHCcons. However, the precision of the10mer predictions are slightly lower in the 0 -150 nM and 0 - 150* nMranges and significantly lower in the 151-500 nM range, compared to the9mers.

TABLE 2 Range (nM) 10mer PAN 10mer netMHC 10mer CONS 0-150 53% (19) 50%(16) 59% (17) 0-150* 68% (19) 69% (16) 76% (17) 151-500 35% (26) 42%(12) 35% (23) FN (>500) 11% (18) 23% (35) 13% (23)

For 10mers, only predictions in the 0 - 150 nM range is utilized due tothe lower than 50% precision for binders in the 151-500 nM range.

The number of samples for any individual HLA allele was too small todraw any conclusions regarding accuracy of the prediction algorithm fordifferent alleles. Data from the largest available subset (0 - 150* nM;9mer) is shown in Table 3 as an example.

TABLE 3 Allele Fraction correct A0101 2/2 A0201 9/11 A0301 5/5 A1101 4/4A2402 0/0 A6801 ¾ B0702 4/4 B0801 ½ B1501 2/2

Only predictions for HLA A and B alleles are utilized as there is littleavailable data on which to judge accuracy of predictions for HLA Calleles (Zhang et al).

An evaluation of melanoma sequence information and peptide bindingpredictions was conducted using information from the TCGA database.Information for 220 melanomas from different patients revealed that onaverage there were approximately 450 missense and 5 neoORFs per patient.20 patients were selected at random and the predicted binding affinitieswere calculated for all the missense and neoORF mutations using netMHC(Lundegaard et al Prediction of epitopes using neural network basedmethods J Immunol Methods 374:26 (2011)). As the HLA allotypes wereunknown for these patients, the number of predicted binding peptides perallotype were adjusted based on the frequency of that allotype (BoneMarrow Registry dataset for the expected affected dominant population inthe geographic area [Caucasian for melanoma]) to generate a predictednumber of actionable mutant epitopes per patient. For each of thesemutant epitopes (MUT), the corresponding native (NAT) epitope bindingwas also predicted.

Utilizing the Prioritization Described Herein

-   90% (18 of 20) of patients were predicted to have at least 20    peptides appropriate for vaccination;-   For nearly a quarter of the patients, neoORF peptides could    constitute half to all of the 20 peptides;-   For just over half of the patients, only peptides in categories 1    and 2 would be used;-   For 80% of the patients, only peptides in categories 1, 2, and 3    would be utilized.

Thus, there is a sufficient number of mutations in melanoma to expect ahigh proportion of patients to generate an adequate number ofimmunogenic peptides.

Example 7 Peptide Production and Formulation

GMP ncoantigenic peptides for immunization is prepared by chemicalsynthesis Merrifield RB: Solid phase peptide synthesis. I. The synthesisof a tetrapeptide. J. Am. Chem. Soc. 85:2149-54, 1963) in accordancewith FDA regulations. Three development runs have been conducted of 20~20-30mer peptides each. Each run was conducted in the same facility andutilized the same equipment as is used for the GMP runs, utilizing draftGMP batch records. Each run successfully produced > 50 mg of eachpeptide, which were tested by all currently planned release tests (e.g.,Appearance, Identify by MS, Purity by RP-HPLC, Content by ElementalNitrogen, and TFA content by RP-HPLC) and met the targeted specificationwhere appropriate. The products were also produced within the timeframeanticipated for this part of the process (approximately 4 weeks). Thelyophilized bulk peptides were placed on a long term stability study andis evaluated at various time points up to 12 months.

Material from these runs has been used to test the planned dissolutionand mixing approach. Briefly, each peptide is dissolved at highconcentration (50 mg/ml) in 100% DMSO and diluted to 2 mg/ml in anaqueous solvent. Initially, it was anticipated that PBS would be used asa diluent, however, a salting out of a small number of peptides caused avisible cloudiness. D5W (5% dextrose in water) was shown to be much moreeffective; 37 of 40 peptides were successfully diluted to a clearsolution. The only problematic peptides are very hydrophobic peptides.

Table 4 shows the results of solubility evaluations of 60 potentialneoantigen peptides, sorted based on the calculated fraction ofhydrophobic amino acids. As shown, almost all peptides with ahydrophobic fraction lower than 0.4 are soluble in DMSO/D5W, but anumber of peptides with a hydrophobic fraction greater than or equal to0.4 were not soluble in DMSO/D5W (indicated by red highlighting in thecolumn labeled “Solubility in DMSO/D5W”). A number of these can besolubilized by addition of succinate (indicated by green highlighting inthe column “Solubility in DMSO/D5W/Succinate”). 3 of 4 of these peptideshad hydrophobic fractions between 0.4 and 0.43. Four peptides becameless soluble upon addition of succinate; 3 of 4 of these peptides had ahydrophobic fraction greater than or equal to 0.45.

Table 4

The predicted biochemical properties of planned immunizing peptides areevaluated and synthesis plans may be altered accordingly (using ashorter peptide, shifting the region to be synthesized in the N- orC-terminal direction around the predicted epitope, or potentiallyutilizing an alternate peptide) in order to limit the number of peptideswith a high hydrophobic fraction.

Ten separate peptides in DMSO/D5W were subjected to two freeze/thawcycles and showed full recovery. Two individual peptides were dissolvedin DMSO/D5W and placed on stability at two temperatures (-20° C. and-80° C.). These peptides were evaluated (RP-HPLC and pH and visualinspection) for up to 24 weeks. Both peptides are stable for up to 24weeks; the percent impurities detected by the RP-HPLC assay did notchange significantly for either peptide when stored at either -20° C. or-80° C. Any small changes appear to be due to assay variability as notrends were noted to be evaluated.

As shown in FIG. 6 , the design of the dosage form process are toprepare 4 pools of patient-specific peptides consisting of 5 peptideseach. A RP-HPLC assay has been prepared and qualified to evaluate thesepeptide mixes. This assay achieves good resolution of multiple peptideswithin a single mix and can also be used to quantitate individualpeptides.

Membrane filtration (0.2 µm pore size) is used to reduce bioburden andconduct final filter sterilization. Four different appropriately sizedfilter types were initially evaluated and the Pall, PES filter (# 4612)was selected. To date, 4 different mixtures of 5 different peptides eachhave been prepared and individually filtered sequentially through twoPES filters. Recovery of each individual peptide was evaluated utilizingthe RP-HPLC assay. For 18 of the 20 peptides, the recovery after twofiltrations was >90%. For two highly hydrophobic peptides, the recoverywas below 60% when evaluated at small scale but were nearly fullyrecovered (87 and 97%) at scale. As stated herein, approaches areundertaken to limit the hydrophobic nature of the sequences selected.

A peptide pool (Pool 4) consisting of five peptides was prepared bydissolution in DMSO, dilution with D5W/Suecinate (5 mM) to 2 mg/ml andpooling to a final peptide concentration of 400 pg per ml and a finalDMSO concentration of 4%. After preparation, peptides were filtered witha 25 mm Pall PES filter (Cat # 4612) and dispensed into Nunc Cryo vials(# 375418) in one ml aliquots. Samples were analyzed at time zero and at2 and 4 weeks to date. Additional samples are analyzed at 8 and 24weeks. At -80° C., no significant change in the HPLC profiles orimpurity profile of the peptide Pool 4 was observed at the four-weektime point. Through the 4 week time point, visual observation and pH forthe peptide pool did not change.

Example 8 Peptide Synthesis

GMP peptides are synthesized by standard solid phase synthetic peptidechemistry (e.g., using CS 536 XT peptide synthesizers) and purified byRP-HPLC. Each individual peptide is analyzed by a variety of qualifiedassays to assess appearance (visual), purity (RP-HPLC), identity (bymass spectrometry), quantity (elemental nitrogen), and trifluoroacetatecounterion (RP-HPLC) and released.

The personalized neoantigen peptides may be comprised of up to 20distinct peptides unique to each patient. Each peptide may be a linearpolymer of --d′.0 - ~30 L-amino acids joined by standard peptide bonds.The amino terminus may be a primary amine (NH2-) and the carboxyterminus is a carbonyl group (—COOH). The standard 20 amino acidscommonly found in mammalian cells are utilized (alanine, arginine,asparagine, aspartic acid, cysteine, glutamine, glutamic acid , glycine,histidine, isoleucine, leucine lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine, valine). The molecularweight of each peptide varies based on its length and sequence and iscalculated for each peptide.

Fmoc (9-fluorenylmethoyloxycarbnyl)-N-terminal protected amino acids areutilized for all synthesis reactions. The side chains of the amino acidsare protected by 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl(Pbf), triphenylmethyl (Trt), t-butyloxycarbonyl (Boc) or t-butyl ether(tBu) group as appropriate. All bulk amino acids are dissolved indimethylformamide (DMF). Condensation utilizes the following twocatalyst combinations in separate reactions:

-   Diisopylcarbodiimide/1- Hydroxybenzotriazole (DIC/HOBT)-   Diisoproplyethylamine/2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium    hexafluorophosphate (DIEA/HBTU)

Each amino acid is coupled twice in order to ensure high level ofincorporation. The first coupling utilizes DIC/IIOBT for 2 - 6 hours andthe second coupling utilizes DIEA/FiBTI.; for 1 - 2 hours. Each of thetwo couplings are monitored by UV absorbance and the resin is washedextensively with DMF in between coupling cycles to improve efficiency.After two cycles of coupling, calculated coupling efficiency must be atleast 95% to continue to the next cycle. Further synthesis of anypeptides that do not meet that minimal coupling efficiency is stopped.

After all amino acids have been coupled, the resin is washed twice withDMF and subsequently three times with methanol. The resin is then vacuumdried briefly while still in the reaction vessel and then transferred toa new, tared vessel for vacuum drying (greater than 12 hours) until itis freely flowing. The mass of crude peptide synthesized is determinedby weighing the vessel containing dried resin, subtracting the mass ofthe tared vessel and adjusting for the resin mass. Expected mass yieldsrange from 60% .. 90%. Any synthesis that failed to produce at least 200mg crude peptide is terminated. The dried resin may be stored at 4oC forup to 28 days prior to initiation of cleavage.

The cleavage reaction is conducted in a single room. Prior to transferof the set of patient-specific dried resins from the synthesis room tothe cleavage room, the cleavage room is fully qualified by QA forsynthesis of a new GMP product. Qualification includes line clearanceinspection, verification of GMP suite cleaning, staging of all requiredmaterials and glassware, verification of equipment suitability andlabeling, and verification that all required personnel are properlytrained and qualified to conduct the work and are properly gowned andfree of apparent illness.

Room readiness operations initiates with verification of the equipmentto be used (rotary evaporator, vacuum pump, balance) and inspection ofdocumentation indicating that the equipment has been properly cleanedand calibrated (if appropriate). A complete list of all raw materials(TFA, triisopropylsilane (TIS) and 1,2-ethanedithiol) required is issuedby QA and manufacturing identifies lot number to be utilized, retest orexpiration date and quantity of material dispensed for each day’sreactions.

Cleavage of the peptide chain from the resin and cleavage of the sidechain protecting groups are accomplished under acidic conditions (95%TFA) in the presence of 2 % triisopropylsilane (TIS) and 1%1,2-ethanedithiol as scavengers of acid-generated free-radicals for 3 to4 hours at room temperature.

Resin is separated from free crude peptide by filtration. The finalsolution of released and de-protected peptide undergoes precipitationwith ether and the precipitate is freeze-dried for 12 hours. The yieldof released crude peptide is determined by weighing the freeze-driedpowder and calculating the ratio of released crude peptide/resin-boundpeptide. Expected yields of crude peptide are 200 mg to 1000 mg. Anycleavage reaction that fails to yield at least 200 mg crude peptide isterminated. The crude peptide is then transferred to the purificationsuite.

The purification is conducted in a single room. Prior to transfer of theset of dried crude peptide from the cleavage room to the purificationroom, the purification room is fully qualified by Quality Assurance forsynthesis of a new GMP product. Qualification includes line clearanceinspection, verification of GMP suite cleaning, staging of all requiredmaterials and glassware, verification of equipment suitability andlabeling, and verification that all required personnel are properlytrained and qualified to conduct the work and are properly gowned andfree of apparent illness.

Room readiness operations initiates with verification of the equipmentto be used (preparative Reverse-Phase High-Performance LiquidChromatography [RP-HPLC], balance, analytical Liquid Chromatography/MassSpectrometer (LC/MS), lyophilizer, balance) and inspection ofdocumentation indicating that the equipment has been properly cleanedand calibrated (if appropriate). A complete list of all raw materials(trifluoroacetic acid [TFA], acetonitrile [ACN], water) required isissued by QA and Manufacturing identifies lot number to be utilized,retest or expiration date and quantity of material dispensed for eachday’s reactions.

Purification is initiated by dissolving no more than 200 mg of thefreeze-dried released peptide in ACN. The sample is then further dilutedwith water to 5% -10% ACN. TFA is added to a final concentration of 0.1%. One C:-18 RP-H PLC column (10 cm x 250 cm) is freshly packed prior tothe initiation of each set of patient specific peptides. Columns areextensively washed with 5% acetonitrile containing 0.1% TFA prior toloading patient peptide. Maximum amount of peptide loaded onto a singlecolumn is 200 mgs. Columns are monitored by UV observance at 220 nm.Following loading of single peptide, the sample is allowed to enter thecolumn and column is washed with 5% acetonitrile/0.1% TFA. A 10% - 50%gradient of acetonitrile with 0.1% TFA is used to elute the peptide.Fractions is collected (50 ml each) beginning at the point UV observanceis at 20% above baseline. Fractions continue to be collected until nofurther UV absorbing material is eluting from the column or the gradientis complete. Typically, the main elution peak is separated into 4 to 8fractions.

Each individual fraction is assessed by analytical LC/MS. Analyticalconditions chosen is based on the percent acetonitrile associated withthe peak eluted product. Fractions with the expected mass and puritygreater than or equal to 95% is pooled as peptide product. Typically 2to 4 fractions meet this pooling requirement. The pooled peptide isplaced into a tared jar for freeze-drying and freeze-dried for 24 to 72hours. The mass of lyophilized peptide is determined by determining themass of the jar containing freeze-dried peptide and subtracting the massof the tared jar.

Portions of the freeze-dried peptide is transferred to quality controlfor analysis and disposition. The remaining is stored at -20oC prior tofurther processing.

Any peptides for which none of the fractions meet the requirement of 95%purity is discarded. No reprocessing of RP-UIPL,C fractions can occur.If sufficient unpurified freeze-dried and cleaved peptide is available,a second sample of the peptide may be purified over the column,adjusting the gradient conditions to improve purity of the elutedpeptide.

The column can then be stripped of any remaining peptide by washingextensively with 4 column volumes of 100% ACN/0.1% TFA and thenre-equilibrated with 5% ACN/0.1% TFA prior to loading the next peptide.

Peptides for an individual patient is sequentially processed over thesame column. No more than 25 peptides are processed over a singlecolumn.

Unit operations for drug substance manufacturing thus constitute:

-   Synthesis:    -   Condensation, wash and re-condensation for each amino acid    -   Resin washing and vacuum drying    -   Transfer to the cleavage suite-   Cleavage:    -   Acid cleavage from the resin    -   Separation of released peptide from the resin and peptide        precipitation    -   Transfer to the purification suite-   Purification:    -   Dissolution in acetonitrile and R1′- HPLC purification    -   Freeze-drying of peak fractions for 24 to 72 hours    -   Removal of aliquots for QC testing and storage of remaining        lyophilized product.

Personalized neoantigen peptides may be supplied as a box containing 2ml Nunc Cryo vials with color-coded caps, each vial containingapproximately 1.5 ml of a frozen DMSO/D5W solution containing up to 5peptides at a concentration of 400 ug/ml. There may be 10 - 15 vials foreach of the four groups of peptides. The vials are to be stored at -80°C. until use. Ongoing stability studies support the storage temperatureand time.

Storage and Stability: The personalized neoantigen peptides are storedfrozen at -80oC. The thawed, sterile filtered, in process intermediatesand the final mixture of personalized neoantigen peptides and poly-ICI,Ccan be kept at room temperature but should be used within 4 hours.

Compatibility: The personalized neoantigen peptides are mixed with ⅓volume poly-ICLC just prior to use.

Example 9 NeoVax, a Neoplasia Vaccine, and Nivolumab in PreventingRelapse Following Autologous Stem-Cell Transplantation in Non-Hodgkin’sLymphoma (NHL).

Three exemplary dosing regimens are provided herein. The first two arefocused on improving the activity of Nivolumab, a fully human IgCi4monoclonal antibody developed for the treatment of cancer that acts byblocking ligand activation of the programmed cell death 1 (PCD1)receptor on activated ‘1’ cells, by evaluating the combination of thenovel personalized neoantigen vaccine (also referred to herein as“NeoVax,” and disclosed in U.S. Provisional Application 61/869,721,61/809,406 and 61/913,127, incorporated by reference in their entiretiesherein), and Nivolumab. The third dosing regimen focuses on improvingthe safety and activity profile of Ipilimumab, a monoclonal antibodydirected against cytotoxic T-lymphocyte-associated antigen-4 (CTLA4),either alone or in combination with Nivolumab by reducing Ipilimumaboverall exposure, enabled by a focusing of Ipilimumab co-thcrapy to thetime-frame of the developing the immune response to NeoVax. This reducedexposure is enabled by the combination with an effective vaccine and canbe achieved by either reducing the dose of Ipilimumab or lengthening theperiod between doses or by delivering Ipilimumab subcutaneously near tothe vaccination site and temporally coincident with vaccination. Each isexplained in more detail herein.

Autologous Hematopoietic Stem Cell Transplant (AHSCT) is an effectiveoption for patients suffering relapse after initial induction therapybut long term remission and cures are rarely observed. Recently, basedon the observed expression of programmed cell death 1 ligand (PDL1) onthe surface of diffuse large B-cell lymphoma (DLBCL) and primarymediastinal large B-cell lymphoma (PMLBCL), a study of anti-PD1 antibody(pidilizumab) treatment following AHSCT was conducted (Armand et al.Journal of Clinical Oncology 31:4199 (2013)); incorporated by referencein its entirety herein). That study showed encouraging indications ofbeneficial activity including improved progression free survival inanti-IIDI treated patients compared to other recent clinical trials,changes in immune cell populations consistent with effective reversal ofPD-1 mediated suppression, and an increase in survival of CD4+, CD54RO+memory cells. Nevertheless, improved therapies are needed, particularlyfor patients who demonstrate residual disease after AHSCT.

While anti-PDI therapy may help relieve local immune suppression andover-come T-cell exhaustion or anergy, limitations on the number andspecificity of the existing T-cell population may prevent maximal impactof this effect. Hence, combining anti-PD 1 therapy with an immunestimulating approach, such as a cancer vaccine, may allow anti-PD1 todemonstrate maximal clinical benefit.

NeoVax (a neoplasia vaccine) is a novel personalized cancer vaccineutilizing the exquisitely tumor-specific neoantigens created by thepersonal mutations found in each patient’s tumor (Hacohen et al CancerImmunology Research 1:11 (2013); Heemskerk et al. EMBO Journal 3″: 194(2013); both incorporated by reference herein). These mutations, becausethey result in peptides that are distinct from “self”-peptides, createepitopes that are expected to escape the immune dampening effects ofcentral tolerance. NeoVax vaccine therapy is being developed to createstronger and more durable responses than those characteristic of themultiple native antigen (“Tumor Associated Antigens; TAAs) which haveclinically proven ineffective to date alone, or in combination withcheckpoint blockade inhibition. NeoVax product is disclosed in disclosedin U.S. Provisional Application 61/869,721, 61/809,406 and 61/913,127,incorporated by reference in their entireties herein, and utilizesmultiple (~20) long peptides as immunogens, each representing separatetarget epitopes, and poly-ICLC as adjuvant. Long peptides and polylCLCrepresent the “best in class” delivery system and immune adjuvantrespectively. The first-in-human clinical study with NeoVax is describedon ClinicalTrials.gov (NC:7′ 01970358).

Combining an effective immune stimulating agent with the relief ofimmune suppression would be expected to improve clinical outcome. Thepost-AHSCT setting is characterized by a low volume of residual disease,the infusion of immune cells to a situation of homeostatic expansion,and the absence of any standard relapse-delaying therapy. These featuresprovide a unique opportunity to test the impact of a checkpointinhibitor, such a Nivolumab, plus NeoVax therapy to delay diseaserelapse.

An exploratory two arm study is carried out to compare treatment withNivolumab alone vs Nivolumab and NeoVax in a small number of patients toevaluate safety and to monitor the impact of treatment on ProgressionFree Survival (PFS) and, for any patients with measurable disease at thetime of confirmatory screening, for Objective Response Rate (ORR).Patients are consented prior to initiation of pre-transplantconditioning in order to collect and sequence tumor tissue for vaccinepreparation and is randomized at time of a confirmatory CT screening(approximately 4 weeks prior to of initiation of therapy). Fifteenpatients are included in each agent arm. The results are compared tocomparable patients receiving no additional post-AHSCT at thisinstitution(s). An outline of the study is shown in FIG. 8 .

Therapy can initiate 4 weeks after AHSCT. The schedule for theindividual arms are as follows:

-   Nivolumablhil-   Nivolumab: Vx on days 1, 4, 8, 15, 22 (Weeks 1, 2, 3 and 4;    “Priming”) and boosts at weeks 11 and 19. Nivolumab (3 mg/kg)    beginning at week 5 and continuing each 3 weeks thereafter with a    last dose at week 23.

Patients on the vaccine alone arm with progressive disease may, at thediscretion of the investigator and the PI, begin receiving Nivolumab atabout 3 weeks to attempt rescue.

CT scans are conducted every 4 months after initiation of therapy (withconfirmatory PET scans at the discretion of the treating physician) witha final CT scan at 16 months after initiation of therapy. All patientsreceiving NeoVax is evaluated for immune responses to the immunizingpeptides.

This study can provide additional safety information in this setting butmost importantly extend and improve, with NeoVax, the encouragingresults observed already with pidilizumab in this setting.

Example 10 NeoVax and Nivolumab in Metastatic Clear Cell Renal CellCarcinoma and Melanoma.

Clear cell Renal Cell Carcinoma (ccRCC) and metastatic melanoma are bothtumor types that have been shown to be responsive to immune modulatingtherapies, including cytokines, vaccines and inhibition of checkpointblockade. Multiple immune modulating therapies have been approved forboth diseases, including Ipilimumab for metastatic melanoma. Nivolumabhas been evaluated as a single agent in non-blinded, non-randomizedPhase 1/11 trial with encouraging results in both diseases and multiplepivotal studies are currently underway utilizing Nivolumab alone orNivolumab and Ipilimumab combinations.

Despite this success of checkpoint blockade, there are still manypatients who do not respond or do not respond robustly and the lack ofcoincident targeted immune stimulation with an effective vaccine mayprevent the maximal impact of such therapy. Checkpoint blockade therapyalone may help relieve local immune suppression and over-come T-cellexhaustion or anergy, but may be limited by the number and specificityof the existing T-cell population- those ‘1’ cells arising from thenormal physiological presentation of the evolving tumor to the hostimmune system. Indeed, in many animal studies, including the studiessupporting the initial development of Ipilimumab, anti-CTLA4 treatmentalone was only mildly effective but was considerably more effective whencombined with a vaccine.

Combining an effective vaccine with the relief of immune suppression mayqualitatively broaden the repertoire of T cell targets as well asstrengthen the activity of extant and newly induced T cells and couldthus significantly improve clinical outcomes.

Separate studies are carried out combining NeoVax with Nivolumab inpreviously untreated metastatic melanoma patients (with a surgicallyaccessible tumor for sequence analysis) and in previously untreatedmetastatic clear cell Renal Cell Carcinoma patients who have aresectable primary tumor in place. An outline of the studies are shownin FIG. 9 . Each study is a two-arm study comparing Nivolumab alone toNivolumab + NeoVax. Patients are consented prior to surgery. Patientsare evaluated for Objective Response Rate (ORR), Progression FreeSurvival (PFS) and Overall Survival (OS). Nivolumab isgin dosing at thestandard single agent dosing level of 3 mg-kg as soon as acceptablefollowing surgical tumor removal and continue each 2 weeks untildocumented progression, discontinuation due to toxicity or withdrawal ofconsent. NeoVax therapy can initiate approximately 8 weeks after tumorresection is administered on days 1, 4, 8, 15, and 22 (Weeks 1, 2, 3 and4; “Priming Phase”) and on weeks 11 and 19 (“boosts”). Nivolumab iswithheld during the period of priming vaccinations. 15 patients areincluded in each arm.

Upon initial indication of progressive disease, patients may, at thediscretion of the investigator, continue on therapy or on the protocoluntil confirmation or not of disease progression (which must be resolvedwithin 6 weeks of initial indication).

CT scans are conducted every 4 months after initiation of therapy (withconfirmatory PET scans at the discretion of the treating physician) witha final CT scan at 24 months after initiation of therapy. All patientsreceiving NeoVax is evaluated for immune responses to the immunizingpeptides.

An effective vaccine provides the opportunity to considerably improvethe clinical outcome of Nivolumab therapy by increasing the breadth ofT-cell responses and NeoVax is a first-in-class vaccine focused onneoantigens, a class of highly personal-tumor specific epitopes notsubject to the immune dampening effects of self-tolerance. Reciprocally,checkpoint blockade inhibition during vaccination may increase thebreadth and immune response level to NeoVax. These exploratory studiesare done in disease settings different than the ongoing pivotal studies(prior to failure with Ipilimumab for Melanoma and prior to systemicanti-VEGF targeted therapy for RCC) allowing collection of safety andefficacy data of Nivolumab alone in these settings.

Example 11 Reduced Dose/Schedule Ipilimumab in Combination With NeoVaxin High Risk Melanoma (Without Nivolumab) and Metastatic Melanoma (WithSystemic Nivolumab).

C′TLA4 was initially identified as negative regulator on the surface ofT-cells that was upregulated shortly after initiation of a de novoimmune response or stimulation of an existing response in order todampen the subsequent immune T-cell response and prevent auto-immunityor uncontrolled inflammation. Thus, the magnitude of the developingimmune response has been closely tied to CTLA4 action. Therapy withanti-CTLA4 antibodies, such as Ipilimumab, was expected to increase theanti-cancer response by blocking the negative regulatory signal andallowing more extensive T-cell expansion. Although many animal and humancorrelative studies have suggested other possible mechanisms of action,the clinical responses observed with Ipilimumab are consistent with sucha hypothesis and recently it was shown that antigen-specific T-cells toa personal neoantigen in a patient treated with Ipilimumab were observedprior to Ipilimumab therapy and increased following therapy (van Root)et al, Journal of Clinical Oncology 31 :e439 (2013)).

Many studies in animals have shown that combining anti-CTLA4 treatmentwith a vaccine enhances, sometimes dramatically, the anti-cancer effectand anecdotal information in humans suggests the same (Hodi et al, PNASUSA 105:3005-3010, 2008; Hodi et al., PNAS USA 100:4712-7; Le et al, JImmunother 36:382-9,2013). This may depend critically on the antigen asthe most dramatic effects of the combination in animals and the effectsin humans are typically observed with complex vaccines such asautologous cellular vaccines and were not observed with a standardindividual tumor associated antigen (such as gp100 for melanoma).Autologous tumor cell vaccines contain both neoantigens and tumorassociated antigens (Hodi et al, 2008 and 2003). In the study of Le etal. (2013), a mixture of two pancreatic allogeneic tumor cell lines wasused as the immunogen. Although it is unlikely there was any overlap ofalmost all neoantigens between the cell lines and the patients,pancreatic cancer is characterized by frequent K-ras mutations atposition 12 which can be immunogenic for multiple HLA types (Weden etal., Int J Cancer 128:1120-8, 2011).

Nivolumab , an anti-Programmed Death Receptor 1 (PD1) antibody, may havea distinct mechanism of action. The ligand for PD1, PDL1, is oftenover-expressed on tumor cells in the native tumor micro-environment andis an inhibitory ligand, causing T-cell anergy/exhaustion of tumorinfiltrating lymphocytes. It seemed likely therefore that a combinationof anti-CTLA4 and anti-PD1 antibodies may yield greater positive resultsbecause they have different mechanisms of action and this has beenobserved in animals (Duraiswamy et al, Cancer Res; 73(12) and possiblyin humans (Wolchok JD et al., N Engl J Med, 2013) . Unfortunately, thecombination therapy appears to be more toxic both in the initial reportand in ongoing clinical evaluation (Wolchok, 2013).

One approach to reducing the apparent toxicity while maintaining orincreasing efficacy of the combination therapy would be to add in aneffective vaccine to the therapeutic regimen (to directly generateeffective de novo T cell responses to additional antigens) andsimultaneously reduce the total exposure to checkpoint blockadeantibodies. Although the two CPB Abs have not been comparedside-by-side, in general anti-CTLA4 appears to generate a highertoxicity profile than anti-PD1, consistent with the effects observed inknock-out mice. Given that CTLA4 has a clear role in the de novo immuneresponse, this would suggest that limiting anti-CTLA4 exposure to thetime frame of antigen exposure may achieve both toxicity reduction andmaintenance of efficacy.

Described herein are four exploratory studies in melanoma patients.Studies 3A and 3B, outlined in FIGS. 10A and 10B, are in metastaticdisease and utilize both Ipilimumab and Nivolumab. Studies 3C and 3D,outlined in FIGS. 10C and 10D, mirror 3A and 3B except that the settingis high-risk disease and do not utilize Nivolumab.

The first study (3A) is a 3 cohort dose escalating (dose escalating forIpilimumab only) study in metastatic melanoma patients. Patients areconsented prior to surgery. Five patients can initially be included ineach cohort. In each cohort, patients can receive Nivolumab beginningjust after surgical excision (for tumor sequencing) at 3 mg/kg (the dosebeing evaluated in all Nivolumab alone phase 3 studies) each 2 weeksuntil NeoVax has been prepared. Nivolumab is with-held during thepriming vaccination phase and during the weeks prior to or during theboost when Ipilimumab is being given. NeoVax therapy can initiateapproximately 8 weeks after tumor resection and is administered on days1, 4, 8, 15, and 22 (Weeks 1, 2, 3 and 4; “Priming Phase”) and on weeks11 and 19 (“boosts”). Nivolumab is resumed in the week following thelast priming dose and in the week following each boost. In the firstcohort, patients can receive Ipilimumab at the start of the primingcycle, one week after the 5th priming dose and prior to each boost, allat a dose of 1 mg/kg. The next cohort can increase the dose associatedwith the start of priming vaccination to 3 mg/kg (the therapeuticallyapproved level) and the dose associated with the end of priming and theboosts can remain at 1 mg/kg. For the third cohort, the start of primingdose and the dose associated with each boost is at 3 mg/kg. (See graphicview of the schedule herein). The maximal exposure to Ipilimumab canthus be three 3 mg/kg doses and one 1 mg/kg dose spread over 19 weeks,about 2.5- fold lower than the approved regimen (four 3 mg/kg dosesspread over 10 weeks). Two expansion cohorts (five patients each) isadded at the highest dose level judged to have a toxicity profilecomparable to that observed with Nivolumab alone and at the next highestdose level (if the overall toxicity profile is acceptable). For eachcohort, the quantitative and qualitative features of the immune responseto the neoantigens are evaluated. Patients are followed for ObjectiveResponse Rate (ORR), Progression Free Survival (PFS) and OverallSurvival (OS).

The second study (3B) is of similar design to study 3A except thatIpilimumab is delivered via subcutaneous injection near to (within 2 cm)each vaccination site with each vaccination in order to focus anti-CTLA4activity on the vaccine draining lymph nodes and limit systemic effects.Nivolumab is delivered as described herein. There is three doseescalation cohorts. The first cohort can inject 0.2 ml of 5 mg/mlIpilimumab at each vaccination site with each vaccination. The secondcohort can increase the volume to 0.5 ml and the third cohort to 1.0 ml.The maximal exposure to Ipilimumab is 140 mg over 19 weeks (about11-fold lower than 840 mg [for a 70 kg patient] spread over 10 weeks forthe approved regimen). Two expansion cohorts (five patients each) isadded at the highest dose level judged to have a toxicity profilecomparable to that observed with Nivolumab alone and at the next highestdose level (if there is one and the overall toxicity profile isacceptable). Patients are evaluated as described herein.

The third and the fourth study, described herein, essentially mirror the1st and 2nd except that each is in high risk disease and does notutilize Nivolumab.

The third study (3C) is a 3 cohort dose escalating (Ipilimumab only)study in high risk (Stage IIIB, IIIC and fully resected IV) melanoma orsurgically resected ccRCC patients. Patients are consented prior tosurgery. Five patients can initially be included in each cohort. NeoVaxtherapy can initiate approximately 8 weeks after tumor resection and isadministered on days 1, 4, 8, 15, and 22 (Weeks 1, 2, 3 and 4; “PrimingPhase”) and on weeks 11 and 19 (“boosts”). In the first cohort, patientscan receive Ipilimumab at the start of the priming cycle, one week afterthe 5th priming dose and prior to each boost, all at a dose of 1 mg/kg.The next cohort can increase the dose associated with the start ofpriming vaccination to 3 mg/kg (the therapeutically approved level) andthe dose associated with the end of priming and the boosts can remain at1 mg/kg. For the third cohort, the start of priming dose and the doseassociated with each boost is at 3 mg/kg. (See graphic view of theschedule herein). The maximal exposure to Ipilimumab can thus be three 3mg/kg doses and one 1 mg/kg dose spread over 19 weeks, about 2.5- foldlower than the approved regimen (four 3 mg/kg doses spread over 10weeks). One expansion cohorts (ten patients) is added at the highestdose level judged to have an acceptable toxicity profile consistent withadjuvant therapy and the recurrence risk profile. For each cohort, thequantitative and qualitative features of the immune response to theneoantigens are evaluated. Patients are followed for Recurrence FreeSurvival (RFS) for up to two years as in protocol 13-240.

The fourth study (3D) is of similar design to Study 3B except thatIpilimumab is delivered via subcutaneous injection near to (within 1 cm)each vaccination site with each vaccination in order to focus anti-CTLA4activity on the vaccine draining lymph nodes and limit systemic effects.There is three dose escalation cohorts. The first cohort can inject 0.2ml of 5 mg/ml Ipilimumab at each vaccination site with each vaccination.The second cohort can increase the volume to 0.5 ml and the third cohortto 1.0 ml. The maximal exposure to Ipilimumab is 140 mg over 19 weeks(about 11-fold lower than 840 mg [for a 70 kg patient] spread over 10weeks for the approved regimen). One expansion cohorts (ten patients) isadded at the highest dose level judged to have an acceptable toxicityprofile consistent with adjuvant therapy and the recurrence riskprofile. For each cohort, the quantitative and qualitative features ofthe immune response to the neoantigens are evaluated. Patients arefollowed for Recurrence Free Survival (RFS) for up to two years as inprotocol 13-240.

The exciting results of the combination of Ipilimumab and Nivolumab istempered by increased toxicity. Although potentially manageable byappropriate monitoring and response (to be confirmed in larger studies),the underlying biology of the molecules and the observations that haveaccumulated from animal and some human studies would suggest thatcoupling an effective vaccine with Ipilimumab may allow a reducedIpilimumab overall exposure while maintaining or increasing efficacy.NeoVax is a paradigm shifting vaccine that has the potential forgenerating significantly stronger and more specific immune responses andto synergize with Ipilimumab. These studies provide the opportunity tomaintain or increase efficacy while reducing toxicity. In addition,extending the reduced intensity concept may allow effective andsetting-appropriate expansion of Ipilimumab to high risk disease.

Example 12 Phase I Study Design Combining NeoVax, a PersonalizedNeoAntigen Cancer Vaccine, With Ipilimumab to Treat High-Risk Renal CellCarcinoma

Sequencing technology has revealed that each tumor contains multiple,patient-specific mutations that alter the protein coding content of agene.₁ Such mutations create altered proteins, ranging from single aminoacid changes (caused by missense mutations) to addition of long regionsof novel amino acid sequence due to frame shifts, read-through oftermination codons or translation of intron regions (novel open readingframe mutations; neoORFs). These mutated proteins are valuable targetsfor the host’s immune response to the tumor as, unlike native proteins,they are not subject to the immune-dampening effects of self-tolerance.Therefore, mutated proteins are more likely to be immunogenic and arealso more specific for the tumor cells compared to normal cells of thepatient.₂

There have been multiple reports indicating that missense mutations orneoORFs in animal tumors induce strong CD8+ cytotoxic T lymphocyte (CTL)reactions, in some cases leading to prevention or eradication ofdisease.₃₋₆. More recently Matsushita and colleagues demonstrated that asingle amino acid change in a normal murine structural protein(spectrin-β2) was the dominant target for immune attack against amethylcholanthrene-induced transplantable tumor, defining whether thetumor would survive or not following transplantation.₇ Furthermore,spectrin-β2 expression was also dramatically reduced in escape variants,providing a clear mechanistic example of the immunoediting hypothesis.At the same time, DuPage and colleagues made a similar observation usinga small immunogenic neoORF in the mouse (ovalbumin peptide).₈ Both ofthese studies indicate that the immuno-edited neoantigen is thereforesufficient as the target of an effective anti-tumor response.

Correspondingly, many human studies of spontaneous regression andlong-term survival have shown that powerful CD8+ T cell responsesagainst mutated epitopes correlate with good clinical responses ₉ Thesestudies began with the first identification of human immunogenicneoantigens _(10,11) and the seminal study by Lenncrz demonstrating thestrength and durability of neoantigen response compared to nativeprotein responses₁₂, and now have included the observations of anincrease in neoantigen-specific CD8+ T cells in a patient responding toanti-CTLA-4 therapy₁₃ and tumor regression following infusion of a tumorinfiltrating lymphocyte (TIL) population highly enriched in a neoantigendirected CD4+ T cell.₁₄ These observations were made in multiple cancertypes for multiple HLA alleles and have been widely observed in tumorinfiltrating T cell populations_(15,16.) Most of the CD8+ T cellresponses show a high degree of specificity toward the mutated missenseepitope compared to the native epitope, represent a high proportion ofcirculating T cells, and result in cells that are more abundant andactive than CD8+ T cell responses in the same patients directed towardover-expressed native antigens.

Thus, in animals and in humans, immune responses to both discrete,mutated antigens (such as missense mutations) and expansive novelantigens (neoORFs) are observationally correlated with regression andlong-term remission. Extending that correlation among a large set(n=468) of patients found in the Cancer Genome Atlas (TCGA) database, arecent meta-analysis of six tumor types revealed a significant survivaladvantage (hazard ratio = 0.53; p=0.002) for patients with at least onepredicted immunogenic neoepitope compared to patients with no predictedimmunogenic epitopes.₁₇

Three studies in humans have directly assessed the immunotherapeuticpotential of mutated antigens. First, follicular lymphoma ischaracterized by uncontrolled growth of a B cell expressing rearrangedimmunoglobulin. Purification of that rearranged immunoglobulin and useas a vaccine may improve disease-free survival.₁₈ The induced CD8+ Tcells showed reactivity to the rearranged, mutated portion of theimmunoglobulin molecule (the idiotype) and not the germline framework.₁₉Second, a mix of peptides corresponding to the oncogenic proteins of HPV(a neoORF for humans) has been shown to result in significant remissionof premalignant lesions induced by HPV.₂₀₋₂₂ Finally, immunization witha synthetic version of an in-frame junctional deletion variant of theepidermal growth factor receptor (EGFRviii) in two studies inglioblastoma patients, a population known to frequently contain thismutation, provided encouraging phase 2 results._(23,24) Importantly, inboth studies, evaluation of tumors in patients with tumor recurrenceshowed that the recurrent tumors almost uniformly (20 of 23) lostexpression of EGFRviii. This was interpreted as clear evidence ofimmunoediting due to immune pressure against an immunogenic neoantigenin humans.

Most cancer vaccines employing peptides as immunogens have utilized“short” peptides. These peptides are typically 9 - 10 amino acids inlength and capable of direct binding to the HLA molecule on the surfaceof HLA-expressing cells. “Long” peptides, about 20-30 amino acids inlength, have recently been shown to produce a more robust and moredurable immune response_(20,21). Long peptides require internalization,processing and cross-presentation in order to bind to HLA molecules;these functions only occur in professional antigen-presenting cells,such as dendritic cells, which can induce strong T cell responses.

Many studies in humans have demonstrated the safety of peptide vaccines.These include studies with multiple short peptides₂₅ as well as multiplelong peptides, including neoORFs. In particular, four studies have beenconducted with a mixture of 10 overlapping long peptides derived fromp53 ₂₆₋₃₀ and five separate studies with a mixture of 13 long peptidesderived from the oncogenic proteins of HPV._(20-22,31,32) In thesestudies, no toxicity higher than grade 2 was observed and most adverseevents were of limited duration and severity. Additionally, manyheterologous antigen preparations have been tested in humans. Suchpreparations include irradiated cell vaccines, _(33,34) tumor celllysates₃₅ and shed tumor cell line antigens.₃₆ These heterogeneousvaccines contain mutated antigens, in the form of intact proteins,partially degraded intracellular protein, and peptides found on thesurface bound to MHC I. Moreover, they contain over-expressed andselectively-expressed molecules as well as many additional nativeproteins. In addition, purified heat shock protein (HSP) 96 peptidecomplexes have been used as antigen; such complexes also contain manymutated peptides.₃₇ None of these studies have reported significantsafety issues directly attributable to the immunogens of the vaccines.

Toll like receptors (TLRs) are important members of the family ofpattern recognition receptors (PRRs) which recognize conserved motifsshared by many micro-organisms, termed “pathogen-associated molecularpatterns” (PAMPS). Recognition of these “danger signals” activatesmultiple elements of the innate and adaptive immune system. TLRs areexpressed by cells of the innate and adaptive immune systems such asdendritic cells (DCs), macrophages, T and B cells, mast cells, andgranulocytes and are localized in different cellular compartments, suchas the plasma membrane, lysosomes, endosomes, and endolysosomes₃₈.Different TLRs recognize distinct PAMPS. For example, TLR4 is activatedby LPS contained in bacterial cell walls, TLR9 is activated byunmethylated bacterial or viral CpG DNA, and TLR3 is activated by doublestranded RNA₃₉. TLR ligand binding leads to the activation of one ormore intracellular signaling pathways, ultimately resulting in theproduction of many key molecules associated with inflammation andimmunity (particularly the transcription factor NF-κB and the Type-Iinterferons).

TLR mediated DC activation leads to enhanced DC activation,phagocytosis, upregulation of activation and co-stimulation markers suchas CD80, CD83, and CD86, expression of CCR7 allowing migration of DC todraining lymph nodes and facilitating antigen presentation to T cells,as well as increased secretion of cytokines such as type I interferons,IL-12, and IL-6. All of these downstream events are critical for theinduction of an adaptive immune response.

Among the most promising cancer vaccine adjuvants currently in clinicaldevelopment are the TLR9 agonist CpG and the synthetic double-strandedRNA (dsRNA) TLR3 ligand poly-ICLC. In prcclinical studies poly-ICLCappears to be the most potent TLR adjuvant when compared to LPS and CpGdue to its induction of pro-inflammatory cytokines and lack ofstimulation of IL-10, as well as maintenance of high levels ofco-stimulatory molecules in DCs40. Furthermore, poly-ICLC was recentlydirectly compared to CpG in non-human primates (rhesus macaques) asadjuvant for a protein vaccine consisting of human papillomavirus(HPV)16 capsomers. Poly-ICLC was found to be much more effective ininducing HPV specific Th1 immune responses₄₁.

Poly-ICLC is a synthetically prepared double-stranded RNA consisting ofpolyI and polyC strands of average length of about 5000 nucleotides,which has been stabilized to thermal denaturation and hydrolysis byserum nucleases by the addition of polylysine andcarboxymethylcellulose. The compound activates TLR3 and the RNAhelicase-domains of MDA5 and RIG3, both members of the PAMP family,leading to DC and natural killer (NK) cell activation and production ofa “natural mix” of type I interferons, cytokines, and chemokines.Furthermore, poly-ICLC exerts a more direct, broad host-targetedanti-infectious and possibly anti-tumor effect mediated by the twoIFN-inducible nuclear enzyme systems, the 2′5′-OAS and the P1/eIF2akinase, also known as the PKR (4-6), as well as RIG-I helicase and MDA5.

In rodents and non-human primates, poly-ICLC was shown to enhance T cellresponses to viral antigens₄₂₋₄₅, cross-priming, and the induction oftumor-, virus-, and autoantigen-specific CD8+ T-cells₄₆₋₄₈. In a recentstudy in non-human primates, poly-ICLC was found to be essential for thegeneration of antibody responses and T-cell immunity to DC targeted ornon-targeted HIV Gag p24 protein, emphasizing its effectiveness as avaccine adjuvant.

In human subjects, transcriptional analysis of serial whole bloodsamples revealed similar gene expression profiles among 8 healthy humanvolunteers receiving one single s.c. administration of poly-ICLC anddifferential expression of up to 212 genes between these 8 subjectsversus 4 subjects receiving placebo₄₉. Remarkably, comparison of thepoly-ICLC gene expression data to previous data from volunteersimmunized with the highly effective yellow fever vaccine YF17D50 showedthat a large number of transcriptional and signal transduction canonicalpathways, including those of the innate immune system, were similarlyupregulated at peak time points.

Two studies of poly-ICLC in conjunction with long peptides have beenpublished. An immunologic analysis was reported on patients withovarian, fallopian tube, and primary peritoneal cancer in second orthird complete clinical remission who were treated on a phase 1 study ofsubcutaneous vaccination with synthetic overlapping long peptides (OLP)from the cancer testis antigen NY-ESO-1 alone or with Montanide-ISA-51,or with 1.4 mg poly-ICLC and Montanide. The generation ofNY-ESO-1-specific CD4+ and CD8+ T-cell and antibody responses weremarkedly enhanced with the addition of poly-ICLC and Montanide comparedto OLP alone or OLP and Montanide.₅₁ In a second human study, poly-ICLCwas combined with a MUC1 synthetic long peptide in patients withpre-malignant adenomas. Robust antibody responses were detected innearly half the patients which inversely correlated with thepre-existing circulating myeloid derived suppressor cell level.₅₂

Poly-ICLC has also been utilized as an adjuvant for immunization withminimal epitope-loaded patient-derived dendritic cells. Both CD4+ andCD8+ T cell responses to multiple peptides were observed in the majorityof patients.₅₃

Poly-ICLC is the TLR3 agonist formulation most extensively tested inpatients with infectious diseases and in subjects with a variety ofdifferent tumor types. Prior to the availability of recombinantinterferon, poly-ICLC was used clinically at high doses ≥ 6 mg/m2 (about170 µg/kg) in patients with a variety of solid tumors and leukemia.₅₃Fever, often above 40° C., was a common adverse event and the primarydose-limiting factor. Other common adverse events were flu-like symptoms(nausea, vomiting, arthralgia. myalgia and fatigue) and hypotension,thrombocytopenia and leukopenia. Once recombinant interferon becameclinically available, the need to pursue high dose poly-ICLC waseliminated, and it became recognized that lower doses (10 - 50 µg/kg)were highly effective at stimulating host defense and as an immune adjuvant.

By now, more than 400 patients with malignant gliomas have been enteredon 7 clinical trials using low dose (1-2 mg total dose) poly-ICLC eitheras monotherapy or in conjunction with chemotherapy, radiation, orvaccine (Table 5). Furthermore, patients with various other solid tumors(prostate, colorectal, pancreatic, hepatocellular, breast, and ovariancancers), in addition to patients with HIV/AIDS and multiple sclerosishave been treated on more than 10 additional clinical phase I and phaseII studies. Overall, the drug has been well-tolerated across all studiesand spectrum of diseases.

Table 5 Clinical Trials with Low-Dose poly-ICLC Patients Protocol TitlePhase Protocol Location Indication Status N Active Year of initationDosing Schedule Long-term IM poly-ICLC in Malignant Glioma - an OpenPilot Study III Walter Reed Malignant Glioma Closed 67 0 1996 IM 10-50µg/kg 1-3 X week Poly-ICLC in Recurrent Malignant Brain Tumors, an OpenLabel Study III MCV Recurrent Glioma Closed 99 0 2000 IM20 µg/kg 3 Xweek Poly-ICLC in Malignant Pediatric Brain Tumors III L.A. ChildrensHosp Pediatric Glioma Closed 46 0 2002 IM 20 µg/kg 2 X week Poly-ICLCplus Radiation in Glioblastoma II NABTC 2001-05 New Glioblastoma Closed31 0 2006 IM 20 µg/kg 3 X week Poly-ICLC in Recurrent Anaplastic GliomaII NABTC 2001-06 Recurrent Anaplastic Glioma Closed 55 6 2006 IM 20µg/kg 3 X week Poly-ICLC plus Temodar in Newly Diagnosed Glioblastoma IINABTT 2005-01 New Glioblastoma Closed 97 24 2006 IM 20 µg/kg 3 X weekPilot Study of MUC1 Vaccine plus poly-ICLC in Advanced Prostate CancerIII UPMC05-086 Advanced Prostate Cancer Open 25 15 2006 1 M 25 µg/kg 2 Xweek Poly-ICLC plus Dendritic Cell vaccine in Recurrent Gliomas I UPMCRecurrent malignant gliomas Open 25 20 2007 IM 20 mcg/kg Poly-ICLC plusHSP-HPVE7 Vaccine in Cervical Dysplasia I Nventa Cervical DysplasiaClosed 24 5 2007 0.05 - 2 mg IntraTumoral poly-ICLC plus Radiation andTACE in Liver Cancer I UMDNJ Hepatoma, Metastatic pancreatic cancer Open31 1 2007 0.25-2 mg A Randomized Controlled Phase I dose escalationtrial of Nasal Hiltonol in normal volunteers. I NIAID, NIH: Normalvolunteers Closed 56 0 2009 0.25-4 mg IN PSMA and TARP peptide with polyICLC adjuvant in ... Prostate Cancer. I/II Moffit Cancer Center ProstateCancer Open 30 2009 1 mg CDX 1307 vaccine with poly-ICLC in metastaticcancers I Various Metastatic Cancers Closed 20 2009 2 mg IM Poly-ICLCwith glioma associated peptide vaccine I/II UPMC Grade II Gliomas Open20 2009 20 mcg/kg IM 2X Wk MUC1 Hundred-mer and poly-ICLC Vaccine forTriple-Negative Breast Cancer. I/II Case Western Triple negative breastcancer Open 37 2009 50 mg IM MUC1 100-mer and poly-ICLC Vaccine forColonic Polyposis I/II UPMC Colonic Adenoma Open 45 2009 0.5 mg SCHiltonol +NYESO1 Protein Vaccine in Ovarian Cancer I/II MSKCC, LICROvarian Cancer Closed 28 0 2009 1.4 mg SC

Clear cell renal cell carcinoma (ccRCC) is one of the ten most commoncancers and the incidence is increasing. Between 20 and 30% of patientsinitially present with metastatic disease, which is generally incurable,whereas 30% of patients who undergo curative-intent nephrectomyexperience recurrence with distant metastases. Targeted systemic agentssuch as inhibitors of vascular endothelial growth factor (VEGF) ormammalian target of rapamycin (mTOR) have provided significant clinicalbenefit for patients with metastatic disease, but tumor responses arenot durable and most patients relapse and eventually succumb to thediseases₅₄. Combinations of existing therapeutic modalities are beinginvestigated in these settings but are associated with significanttoxicities and modest added activity. Effective adjuvant treatment ofresected, high-risk patients with presumably micrometastatic, if any,disease burden is lacking. Innovative approaches are therefore necessaryto achieve substantial improvement.

Perhaps more than any other therapeutic approach, immunotherapy has thepotential for curative outcome by capturing the diversity andlong-lasting memory of the immune system and its ability to destroymalignant tumor cells and prevent relapse. High dose interleukin-2(HD-IL2) is used in patients with metastatic ccRCC based on a durablecomplete remission rate of 7%, but this therapy is only offered inselect centers due to its significant toxicity and there is noestablished biomarker to predict for response₅₅. Nevertheless, theresults with HD-IL2 provide proof that “first generation” immunotherapyattempts can work in ccRCC.

Two immunotherapeutic approaches are being actively evaluated in ccRCCand other diseases - antigen-specific immunization (vaccination) and,more recently, non-specific immune stimulation via treatment withcheckpoint blockade antibodies (CPB)₅₆₋₅₈. Vaccines in ccRCC to datehave provided hints of efficacy but tumor response rates and durabilityof tumor responses remain low,_(59,60) most likely due to the lack ofeffective tumor-specific immune responses induced with these approaches.Historically, most cancer vaccines have utilized tumor-associatedantigens (TAA), a class of native proteins, which are preferentially orselectively expressed on tumor cells, but can also be found on somenormal cells. Native proteins generate relatively weak immune responsesdue to central tolerance (the naturally occurring phenomenon thatprevents the immune system from recognizing self-antigens and therebygenerating auto-immunity). Despite the relatively weak results achievedwith TAA vaccination to date, several pivotal clinical studies areunderway in ccRCC utilizing such native antigens, pointing to the demandfor more effective therapies.

DNA sequencing, particularly next-generation sequencing technology, hasrevealed a genetic landscape of cancer that contains many protein codingmutations found uniquely in the tumor of an individual patient. Thesemutations include both single-amino acid missense mutations (thepredominant type) and novel open reading frames created by frame-shiftor read-through mutations (neoORFs) varying in length from 1 to up to100′s of amino acids. There is evidence in both animals and humansdemonstrating that such mutated epitopes are effective at inducing animmune response. Importantly, strong CD8+ T cell responses againstmutated epitopes have been found in patients with multiple tumor typeswho either had a spontaneous regression or a dramatic response afteradoptive T cell transfer, suggesting that there may be an associationwith CD8+ T cell activity and good clinical responses₉. Most of theseCD8+ T cell responses showed very good specificity toward the mutatedepitope compared to the native epitope, represented a high proportion ofcirculating T cells, and induced cells that were more abundant andactive than CD8+ T cells in the same patients directed towardover-expressed native antigens.

Three studies in humans have directly assessed the immunotherapeuticpotential of mutated antigens. Purified rearranged immunoglobulinexpressed by malignant B cells in follicular lymphoma has been used asvaccine and has led to good clinical outcomes in some phase 2 and phase3 studies (possibly dependent on amounts of residual disease)₆₁. Morerecently, a mix of peptides corresponding to the oncogenic proteins ofHPV (directly analogous to the neoORF type of mutation) has been shownto result in significant remission of premalignant lesions induced byHPV₂₀₋₂₂. Finally, a mutated form of the epidermal growth factorreceptor (EGFR) commonly found in glioblastoma patients has been used asan immunogen in multiple early clinical trials.₆₂ Interestingly,evidence for down-regulation of this mutated gene has been found,suggesting immune editing due to immune pressure induced by thevaccine.₂₄ Thus, in animals and in humans, immune responses to bothdiscrete mutated antigens (such as missense mutations) and expansivenovel antigen (neoORF) are observationally correlated with regressionand long-term remission and, in three clinical studies, have been shownto control disease following therapeutic vaccination. The direct andcomprehensive identification of the many mutated epitopes found incancer genomes creates the opportunity to use this class of immunogen toimprove the immune response and efficacy of cancer vaccines.

Ipilimumab, an anti-CTLA4 specific IgG1 monoclonal antibody, blocks akey regulatory pathway that dampens both de novo and memory T-cellresponses following antigen activation and has been demonstrated toimprove overall survival as monotherapy in patients with advancedmelanoma₅₆. Furthermore, antibodies targeting the PD-1 pathway (anotherimmune checkpoint) have demonstrated impressive anti-tumor activity inmultiple cancer types_(57,58,63-65) and the combination of CTLA-4 andPD-1 pathway blockade showed apparent synergy in advancedmelanoma_(63,64) and most recently in ccRCC (Hammers et al, ASCO 2014Abstract #4504). Despite this success, the lack of coincident immunestimulation with an effective vaccine may limit the maximal impact ofsuch therapy.

Many studies in animals have shown that combining anti-CTLA4 treatmentwith a vaccine can substantially augment efficacy ₆₆₋₆₈ and anecdotalinformation suggests a similar effect in humans _(69,70). This potentialsynergy may depend critically on the antigen as the most dramaticeffects of the combination in animals and the effects in humans havetypically been observed with complex vaccines such as autologouscellular vaccines and not with an individual tumor-associated antigen(such as gp100 for melanoma). Autologous tumor cell vaccines ₆₉ containboth neoantigens and tumor-associated antigens. NeoVax, by focusing theimmune system on neoantigens, presumably the most immunogenic subset ofpossible antigens, is expected to strengthen and focus the immuneresponse to the tumor, thereby generating more robust clinical activity.Reciprocally, checkpoint blockade during vaccination may increase thebreadth and the magnitude of the response to NeoVax and increase memorycell formation. Recently, neoantigen-specific T-cells were observed inan Ipilimumab-treated patient prior to treatment initiation andincreased following therapy ₇₁.

An innovative aspect of the study is the route of delivery ofIpilimumab. There are real concerns regarding the toxicity profile ofintravenously delivered Ipilimumab, the approved delivery route formetastatic disease. For this reason, the study design includes deliveryof Ipilimumab “locally” by sub-cutaneous injection in proximity to thevaccination site during each vaccine dosing. Large proteins likeIpilimumab enter the circulation following sub-cutaneous injection bytransit through the lymphatics and draining lymph nodes₇₂. Thus, it isexpected that this approach can target Ipilimumab to the same draininglymph node as the vaccine and reduce systemic exposure, thereby bothmaximizing effectiveness and limiting systemic toxicity of Ipilimumab.Multiple animal studies have shown that localized dosing of anti-CTLA4is effective. The modes of localized dosing have included: (i) localproduction of anti-CTLA4 from the irradiated tumor cell being used asvaccine ₇₃, (ii) “in transit” delivery of anti-CTLA4 between the tumor(antigen source) and the local draining lymph node ₇₄, and (iii) directintra-tumoral injection of anti-CTLA4₇₅. Anti-tumor activity withlocalized administration from irradiated anti-CTLA4 expressing tumorcells was reduced compared to systemic administration, but it was equalor better with “in transit” delivery and with direct intra-tumoralinjection. Of note, an abscopal effect was observed, wherein injectionof anti-CTLA4 antibody near to or into a tumor on one flank of theanimal resulted in elimination of the tumor on the opposite flank_(74,75) indicating a systemic treatment effect.

Described herein is a study design combining a personalized neoantigencancer vaccine with Ipilimumab to treat high-risk renal cell carcinoma(see FIG. 11 ). The study is an open label, phase I trial in whichpatients with clear ccRCC (both fully resected but high risk patients aswell as patients with low or intermediate risk metastatic disease) isimmunized with up to 20 peptides that are both specific to theparticipant’s tumor cells (i.e. - not found in their normal cells) andunique to the participant (i.e. - “personal”) and concurrently receiveIpilimumab in close proximity to the vaccine site. These peptides areencoded by missense mutations, in-frame gene fusions and novel openreading frame mutations (collectively known as “neoantigens”) that haveoccurred within that participant’s tumor cells and are identifiedthrough DNA and RNA sequencing. Up to 20 peptides at least ~20 aminoacids in length is prepared for each participant and is administeredtogether with the immune adjuvant polyinosinic-polycytidylic (poly-IC)stabilized with poly-L-lysine and carboxymethylcellulose(poly-ICLC)(Hiltonol ®). Thus, the personalized NeoAntigen CancerVaccine consists of peptides + poly-ICLC and is termed “NeoVax”.Ipilimumab is an antibody against CTLA4, a molecule on the surface of Tcells that limits T cell expansion, and has been approved for thetreatment of metastatic melanoma (Yervoy®). Ipilimumab is delivered viasubcutaneous injection in proximity to each vaccination site in orderto 1) direct anti-CTLA4 activity to the vaccine-draining lymph nodes and2) limit systemic toxic effects. This approach is effective in animaltumor models and is expected to result in significantly reduced levelsof systemic Ipilimumab compared to the approved dose/schedule inadvanced melanoma (3 mg/kg q3wks for four doses).

Eligible patients are entered into the trial to undergo surgery with theintent to resect the primary kidney tumor or a metastatic site. Patientswho have undergone surgery, fulfill eligibility criteria, and for whomsufficient tissue was obtained to prepare nucleic acid for sequencingcan continue onto the treatment phase of the trial.

The study is conducted in two parts. In the first part (10 patients) themaximum tolerated dose (MTD) is identified from three possible doselevels. In the second part, 10 additional patients are enrolled at theMTD to extend the safety and activity analysis of the NeoVax cancervaccine given in combination with locally administered ipilimumab.

Five patients are entered for the initial safety evaluation (Cohort 1).If none or only 1 patient experiences a DLT during the first 7 weeks oftreatment of Cohort 1, 5 patients are entered to Cohort 2. If two ormore patients in Cohort 1 experience dose limiting toxicity (DLT) duringthe first 7 weeks of treatment, then 5 patients are entered on Cohort-1.

If none or only 1 patient experiences a DLT on Cohort 2, then Dose Level2 is the maximum tolerated dose (MTD) and an additional 10 patients areentered at that dose level to increase the likelihood of detectingserious toxicities, to complete biologic correlative endpoints and togain preliminary experience with clinical tumor activity. If two or morepatients in Cohort 2 experience dose limiting toxicity (DLT), then DoseLevel 1 is the MTD and an additional 10 patients are treated at thisdose.

If none or only 1 patient experiences a DLT on Cohort -1, then DoseLevel-1 is the maximum tolerated dose (MTD) and an additional 10patients are entered at that dose level. If two or more patients inCohort 2 experience dose limiting toxicity (DLT), then the study isstopped.

GMP peptides are prepared by synthetic chemistry, purified by ReversePhase High-Performance Liquid Chromatography (RP-HPLC), mixed in smallgroups and combined with the immune adjuvant poly-ICLC, a stabilizeddouble-stranded RNA. The mixtures of peptides and poly-ICLC is used forvaccination with the intention to induce cellular immune responsesdirected at these patient/tumor specific mutations. Each participant canreceive the full complement of peptides at each immunization.

The induction of a ncoantigcn-spccific T cell response followingvaccination with NeoVax and simultaneous treatment with locallydelivered Ipilimumab is assessed by IFN-γ EUSPOT and/or tetrameranalysis. Comparison is made between samples taken prior to vaccineadministration and after vaccination, beginning 4 weeks after the lastpriming dose. Assays are conducted for all 10 patients in the expansioncohort only.

IFN-γ secretion occurs as a result of the recognition of cognatepeptides or mitogenic stimuli by CD4+ and/or CD8+ T -cells. A multitudeof different CD4+ and CD8+ determinants can be presented to T cells invivo since the 20-30-mer peptides used for vaccination should undergoprocessing into smaller peptides by antigen presenting cells. Patientsare evaluated using the predicted epitope short peptides as well aslonger peptides which require proteasomal processing as stimulant in theIFN-γ ELISPOT assay. If warranted, the precise immunogenic peptide(s) isdetermined in follow-up analyses.

When feasible, HLA-tetramers are prepared for one or more epitopes andis used for cell staining and flow cytometry to independently evaluatethe level of responding T cells.

In addition to the analysis of the magnitude and determinant mapping ofthe T cell response in peripheral blood, other aspects of the immuneresponse induced by the vaccine are critical and is assessed. Theseevaluations are performed in patients who exhibit an ex vivo IFN-yELISPOT or tetramer response in the screening assay. They include theevaluation of T cell subsets (Th1 versus Th2, T effector versus memorycells), analysis of the presence and abundance of regulatory cells suchas T regulatory cells or myeloid derived suppressor cells, andpatient-specific tumor cell recognition. Finally, through targeted deepsequencing of the Vβ subfamily of the T cell receptor in peripheralblood samples collected before and after vaccination or in TILpopulations, the global changes in TCR repertoire as well as changes inthe abundance of individual T cell clones are determined.

After adequate tumor for pathological assessment has been harvested,remaining tumor tissue is placed in sterile media in a sterile containerand transferred for immediate freezing or is used for disaggregation.Portions of the tumor tissue is used for whole-exome and transcriptomesequencing. If single cells are prepared, a cell line may be initiated.Additionally, tumor infiltrating lymphocyte is prepared from the singlecell suspension. In the event that sequence analysis yields no resultsor sub-optimal results, tumor cell line cells may be used to prepareadditional nucleic acid for sequencing. Peripheral blood mononuclearcells from a blood draw is utilized for a normal tissue sample.

Nucleic acid is extracted from the tissue samples and sequencing isconducted at the CLIA-certified laboratory at Broad Institute. For tumorand normal DNA samples, whole exome capture is conducted prior tosequencing on Illumina HiSeq. For tumor RNA, a cDNA library is preparedon poly-A selected RNA prior to sequencing on Illumina HiSeq. If thequantity or quality of DNA or RNA isolated from the tissue sample isinadequate for exome or cDNA library preparation and sequencing, thenDNA or RNA may be extracted from the patient-specific tumor cell line(if generated).

Whole exome DNA sequence of tumor and normal tissue samples from thepatient is used to identify the specific coding-sequence mutations thathave occurred in the tumor of that participant. These mutations includeboth single-amino acid missense mutations (the predominant type ofmutation) and novel open reading frames (neoORFs) varying in length fromone up to hundreds of amino acids. A well-established algorithm(netMHCpan) is used to identify mutation-containing epitopes that arepredicted to bind to the MHC class I molecules of each participant.₇₆From this list of candidate mutations, 20 - 40 mutations are selectedand prioritized for peptide preparation based on a pre-defined set ofcriteria including:

-   Type of mutation (missense vs neoORF)-   Predicted binding potential of peptides encoded by the mutated    region to the MHC class I alleles of the particular individual-   Predicted binding potential of the corresponding native peptide-   The likelihood that the mutation is directly or indirectly related    to the tumorigenic phenotype (i.e. an “oncogenic driver” mutation or    a mutation in a related biochemical pathway)-   RNA expression-   Biochemical properties of the full peptide (e.g. predicted poor    solubility secondary to hydrophobic amino acid number or    distribution and/or cysteine content).

Twenty to forty mutations for each participant is used to designpeptides, each approximately 20 - 30 amino acids in length. Analysis ofmutations is limited to comparison of normal and tumor sequenceinformation for each participant.

GMP peptides are synthesized by standard solid phase synthetic peptidechemistry and purified by RP-HPLC. Each individual peptide is analyzedby a variety of qualified assays to assess appearance (visual), purity(RP-HPLC), identity (by mass spectrometry), quantity (elementalnitrogen), and trifluoro-acetate counterion (RP-HPLC) and released. Thiswork is performed by CS Bio, Menlo Park, CA. Synthesis are initiatedwith up to 25 peptides if possible so that additional peptides areimmediately available for replacement of insoluble peptides if needed.

It is intended to immunize patients with as many peptides as possible,up to a maximum of 20. Peptides are mixed together in 4 pools of up to 5peptides each. The selection criteria for each pool is based on theparticular MHC allele to which the peptide is predicted to bind.Peptides predicted to bind to the same MHC allele is placed intoseparate pools whenever possible in order to limit antigeniccompetition. Some of the neoORF peptides may not be predicted to bind toany MHC allele of the patient. These peptides can still be utilizedhowever, primarily because they are completely novel and therefore notsubject to the immune-dampening effects of central tolerance, thushaving a high probability of being immunogenic. NeoORF peptides alsocarry a dramatically reduced potential for autoimmunity as there is noequivalent molecule in any normal cell. In addition, there can be falsenegatives arising from the prediction algorithm and it is possible thatthe peptide can contain a HLA class II epitope (HLA class II epitopesare not reliably predicted based on current algorithms). All peptidesnot identified with a particular HLA allele is randomly assigned to theindividual pools.

The peptide pools are prepared. The amounts of each peptide arepredicated on a final dose of 300 µg of each peptide per injection. Thepeptide pools are prepared by dissolving appropriate quantities of eachpeptide individually at high concentration (approximately 50 mg/ml) indimethyl sulfoxide (DMSO) and dilution with 5% dextrose in water (D5W)/5 mM succinate to a final concentration of 2 mg/ml. Any peptides that donot demonstrate clear solutions upon dilution is discarded and replacedwith another peptide if available; if no additional peptides areavailable, D5W/succinate wil be used. Equal quantities of each of 5peptides can then be admixed, effectively diluting each peptide to aconcentration of 400 µg/ml. The bioburden in the pooled peptides arereduced by filtration through a 0.2 µm sterilizing filter. The pooledand filtered bulk is filter sterilized in a laminar flow biosafetycabinet and aliquoted into 2 ml Nunc cryo individual dosing vials forstorage. Each pool is tested by RP-HPLC for identity, residual solvents(by gas-chromatography), sterility and endotoxin. Individual dosingvials are stored frozen at -80° C. .

The standard and approved 10 ml vial (5 mg Ipilimumab/ml) is utilizedfor this clinical study. No additional preparation is required.

Preparation of the final NeoVax product and syringes containingIpilimumab is begun upon confirmation that the patient is medicallycleared to undergo vaccine administration (confirmed arrival in theclinic, stable vital signs, no new acute medical issues or laboratoryabnormalities potentially interfering with vaccine administration).

The final step in the preparation of NeoVax (mixing with poly-ICLC) isconducted on the day of the scheduled vaccine administration. For eachpatient, four distinct pools (labeled “A”, “B”, “C” and “D”) of up to 5synthetic peptides each can be prepared at the GMP peptide manufacturerand filter sterilized as described in detail and stored at -80° C.

On the day of immunization, the complete vaccine consisting of thepeptide component(s) and poly-ICLC is prepared in a laminar flowbiosafety cabinet. One vial each (A, B, C and D) is thawed at roomtemperature in a biosafety cabinet. 0.75 ml of each peptide pool iswithdrawn from the vial into separate syringes. Separately, four 0.25 ml(0.5 mg) aliquots of poly-ICLC is withdrawn into separate syringes. Thecontents of each peptide-pool containing syringe can then be gentlymixed with a 0.25 ml aliquot of poly-ICLC by syringe-to-syringetransfer. The entire one ml of the mixture is used for injection.

These four preparations are labeled “NeoVax A”, “NeoVax B”, “NeoVax C”,“NeoVax D”. The total dose of NeoVax can consist of the four 1 mlsyringes each containing 1 ml of the peptide pool + poly-ICLC mixtures.

On each dosing day, a single 10 ml vial containing 5 mg/ml Ipilimumab isused to prepare 4 syringes, each containing 0.25 ml (Cohort-1), 0.5 ml(Cohort 1) or 1 ml (Cohort 2).

Vaccine is administered following a prime/boost schedule. Priming dosesof vaccine is administered on days 1, 4, 8, 15, and 22 as shown hereinand (FIG. 12 ). In the boost phase, vaccine is administered on days 78(week 12) and 162 (week 20).

Treatment Description Agent Treatment phase Schedule Allowable TreatmentAdmin Window Pre-medications Precautions Dose Route NeoVax (peptides +poly- Prime Day 1 N/A none Poly-ICLC: 4 × 0.5 mg (total dose 2 NeoVaxs.c. injections into 4 different anatomic Days 4 and 8 ± 1 day ICLC) andIpilimuma b Days 15 and 22 ± 3 days but at least 5 days must elapsebetween doses mg) Peptides: 4 × 300 µg per peptide Volume = 4 × 1 mlIpilimumab 4 × 0.25 ml, 0.5 ml or 1.0 ml depending on the cohort sitesIpilimumab s.c injection within 1 cm of each NeoVax injection Boost Days78 and 134 ± 7 days

Each of the 4 NeoVax and Ipilimumab syringes is assigned to one of fourextremities. At each immunization, each NeoVax syringe is administereds.c. to the assigned extremity (i.e. NeoVax A is injected into left armon day 1, 4, 8 etc., NeoVax B is injected into right arm on days 1, 4, 8etc.). Alternative anatomical locations for patients who are status postcomplete axillary or inguinal lymph node dissection or othercontraindications that prevent injections to a particular extremity arethe left and right midriff, respectively.

Immediately following NeoVax administration at a respective extremity(or alternative anatomical location), Ipilimumab is injected within 1 cmof each NeoVax administration.

NeoVax and Ipilimumab may be administered within 1 day of the scheduledadministration date for days 4 and 8, within 3 days of the scheduledadministration date for days 15 and 22 (but must be at least 5 daysapart) and within 7 days for days 78 and 162.

Five patients are entered for the initial safety evaluation (Cohort 1).If none or only 1 patient experiences a DLT during the first 7 weeks oftreatment of Cohort 1, 5 patients are entered to Cohort 2. If two ormore patients in Cohort 1 experience dose limiting toxicity (DLT) duringthe first 7 weeks of treatment, then 5 patients are entered on Cohort-1.

If none or only 1 patient experiences a DLT on Cohort 2, then Dose Level2 is the maximum tolerated dose (MTD) and an additional 10 patients areentered at that dose level to increase the likelihood of detectingserious toxicities, to complete biologic correlative endpoints and togain preliminary experience with clinical tumor activity. If two or morepatients in Cohort 2 experience dose limiting toxicity (DLT), then DoseLevel 1 is the MTD and an additional 10 patients are treated at thisdose.

If none or only 1 patient experiences a DLT on Cohort -1, then DoseLevel-1 is the maximum tolerated dose (MTD) and an additional 10patients are entered at that dose level.

If two or more patients in Cohort 2 experience dose limiting toxicity(DLT), then the study is stopped.

Duration of therapy can depend on tolerability of the immunizations andevidence of disease recurrence. In the absence of treatment delays dueto adverse events, treatment is given until the day 134 vaccination (the2nd booster vaccination) or until one of the following criteria applies:

-   Disease recurrence, if it is deemed by the treating investigator to    be in the best interest of the patient to discontinue study    treatment-   Intercurrent illness that prevents further administration of    treatment-   Unacceptable adverse event(s)-   Patient demonstrates an inability or unwillingness to comply with    protocol requirements-   Patient decides to withdraw from the study, or-   General or specific changes in the patient’s condition which render    the patient unacceptable for further treatment in the opinion of the    treating investigator.

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Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theabove paragraphs is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

1-47. (canceled)
 48. A method of treating a neoplasia in a subject inneed thereof comprising: (A) administering a cell therapy, wherein thecell therapy comprises (1) antigen presenting cells (APCs) comprising:(i) one or more polypeptides comprising at least two peptide sequencescalculated by an HLA peptide binding analysis using a programimplemented on a computer system to have a binding affinity to a proteinencoded by an HLA allele of the subject with an IC50 of less than 500nM, or (ii) one or more polynucleotides encoding the at least twopeptide sequences; or (2) T cells stimulated with APCs comprising (i) or(ii); wherein the at least two peptide sequences are encoded by aplurality of cancer specific nucleic acid sequences that are identifiedas being specific to cancer cells of the subject; wherein the pluralityof cancer specific nucleic acid sequences that are identified as beingspecific to cancer cells of the subject encodes two or more differentpeptide sequences of two or more different proteins that are expressedby the cancer cells; wherein two or more different peptide sequences oftwo or more different proteins comprise a cancer-specific amino acidmutation that is not present in non-cancer cells from the subject, and(B) administering at least one checkpoint inhibitor, wherein the atleast one checkpoint inhibitor is nivolumab or pembrolizumab.
 49. Themethod of claim 48, wherein the at least two peptide sequences comprisesat least three, at least four or at least five peptides sequences. 50.The method of claim 48, wherein the at least two peptide sequencesranges from 5 to 50 amino acids in length.
 51. The method of claim 48,wherein (i) the subject is suffering from a neoplasia selected from thegroup consisting of: Non-Hodgkin’s Lymphoma (NHL), clear cell Renal CellCarcinoma (ccRCC), melanoma, sarcoma, leukemia or a cancer of thebladder, colon, brain, breast, head and neck, endometrium, lung, ovary,pancreas or prostate,; (ii) the subject has no detectable neoplasia butis at high risk for disease recurrence; or (iii) the subject haspreviously undergone autologous hematopoietic stem cell transplant(AHSCT).
 52. The method of claim 48, wherein the neoplasia ismetastatic.
 53. The method of claim 48, wherein (i) administration ofthe checkpoint inhibitor is initiated before initiation ofadministration of the cell therapy, (ii) administration of thecheckpoint inhibitor is withheld during the week prior to administrationof the cell therapy, or is withheld during administration of the celltherapy; (iii) administration of the checkpoint inhibitor is initiatedafter initiation of administration of the cell therapy; (iv)administration of the checkpoint inhibitor is initiated simultaneouslywith the initiation of administration of the cell therapy, or (iv)administration of the immune checkpoint inhibitor is initiated followingtumor resection.
 54. The method of claim 48, wherein (i) administrationof the checkpoint inhibitor continues every 2-8 or more weeks after thefirst administration of the checkpoint inhibitor, p (ii) administrationof the checkpoint inhibitor continues every 2, 3 or 4, 6 or 8 weeksafter the first administration of the checkpoint inhibitor; (iii)administration of the checkpoint inhibitor is initiated following tumorresection; (iv) administration of the cell therapy is initiated 1-15weeks after tumor resection; or (v) administration of the cell therapyis initiated 4-12 weeks after tumor resection.
 55. The method of claim48, wherein the cell therapy is administered intravenously.
 56. Themethod of claim 48, further comprising administration of one or moreadditional agents, wherein the one or more additional agents (i) areselected from the group consisting of: chemotherapeutic agents,anti-angiogenesis agents and agents that reduce immune-suppression; (ii)are one or more anti-glucocorticoid induced tumor necrosis factor familyreceptor(GITR) agonistic antibodies or (iii) is an anti-CTLA-4 antibody.57. The method of claim 48, wherein the cell therapy comprises APCscomprising (i) or (ii), and the APCs are from the subject.
 58. Themethod of claim 48, wherein the cell therapy comprises T cellsstimulated with APCs comprising (i) or (ii), and the T cells are fromthe subject.
 59. The method of claim 48, wherein the cell therapycomprises the T cells and a pharmaceutically acceptable carrier orexcipient.
 60. The method of claim 48, wherein the cell therapycomprises APCs comprising (i) or (ii), and the APCs are not from thesubject.
 61. The method of claim 48, wherein the cell therapy comprisesT cells stimulated with APCs comprising (i) or (ii), and the T cells arenot from the subject.
 62. The method of claim 48, wherein the bladdercancer is bladder carcinoma.
 63. The method of claim 48, wherein thebladder cancer is a cancer of the renal pelvis or ureter.
 64. The methodof claim 48, wherein the melanoma is metastatic melanoma.
 65. The methodof claim 48, wherein the subject has not been treated previously and hasmetastatic melanoma.
 66. The method of claim 48, wherein the at leasttwo peptide sequences ranges from 15 to 35 amino acids in length. 67.The method of claim 48, wherein one or more polypeptides comprising atleast two peptide sequences calculated by an HLA peptide bindinganalysis using a program implemented on a computer system to have abinding affinity to a protein encoded by an HLA allele of the subjectwith an IC50 of less than 150 nM.
 68. The method of claim 48, whereinthe at least two peptide sequences are identified by a. whole genome orwhole exome nucleic acid sequencing of a nucleic acid sample of thesubject’s tumor and of a non-tumor sample of the subject; and b.identifying at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19 or 20 of non-silent tumor mutations, wherein the mutations arepresent in the genome of cancer cells of the subject but not in normaltissue from the subject, wherein the non-silent mutations are determinedby analysis of the sequences obtained by the whole genome or whole exomenucleic acid sequencing.
 69. The method of claim 48, wherein the atleast one checkpoint inhibitor is administer prior to administering thecell therapy.
 70. The method of claim 48, wherein the subject is notimmuno-compromised by a previous cancer-directed therapy.
 71. The methodof claim 48, wherein the at least one checkpoint inhibitor isadministered intravenously.